JP7228269B2 - Cell mass fusion method - Google Patents

Cell mass fusion method Download PDF

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JP7228269B2
JP7228269B2 JP2020509172A JP2020509172A JP7228269B2 JP 7228269 B2 JP7228269 B2 JP 7228269B2 JP 2020509172 A JP2020509172 A JP 2020509172A JP 2020509172 A JP2020509172 A JP 2020509172A JP 7228269 B2 JP7228269 B2 JP 7228269B2
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英樹 谷口
友美 田所
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Yokohama City University
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Description

本発明は、細胞塊を融合させる方法に関する。 The present invention relates to a method of fusing cell clusters.

生体内のほとんどの組織・臓器には血管系が存在し、血液細胞・酸素・栄養等の供給、老廃物等の除去を行っている。
近年、生体外における細胞培養により、腸や肝臓など様々な上皮系細胞を用いた３次元培養法が確立されている（非特許文献１、２）が、それらは上皮細胞のみからなるオルガノイドであり、血管構造は含まれていない。
このような技術に続き、臓器の前駆細胞と血管内皮細胞、間葉系細胞を混合させ、マトリゲルなどのマトリクス上で自律的な細胞凝集を惹起することにより、微小血管網を有するオルガノイド（臓器原基）を作製する技術が開発されている（非特許文献３、特許文献１）。Most tissues and organs in the living body have a vascular system, which supplies blood cells, oxygen, nutrients, etc., and removes waste products.
In recent years, three-dimensional culture methods using various epithelial cells such as intestinal and liver cells have been established by in vitro cell culture (Non-Patent Documents 1 and 2), but they are organoids consisting only of epithelial cells. , vascular structures are not included.
Following this technique, organ progenitor cells, vascular endothelial cells, and mesenchymal cells were mixed to induce autonomous cell aggregation on matrigel and other matrices, resulting in organoids with microvascular networks (organ progenitor cells). group) has been developed (Non-Patent Document 3, Patent Document 1).

低吸着プレート等を用いることにより、マトリクスフリーで細胞塊を作製することは可能であるが、主に栄養と酸素要求性の問題から内部でネクローシスを起こさずに長期培養を行うには細胞塊を数百μｍ以下の大きさに留める必要がある（非特許文献４）。 Although it is possible to produce matrix-free cell aggregates by using low-adsorption plates, etc., cell aggregates are not recommended for long-term culture without internal necrosis, mainly due to the problem of nutrient and oxygen requirements. It is necessary to limit the size to several hundred μm or less (Non-Patent Document 4).

Nature 459, 262-265, 2009Nature 459, 262-265, 2009 Cell 160, 299-312, 2015Cell 160, 299-312, 2015 Nature 399, 481-484, 2013Nature 399, 481-484, 2013 Br. J. Cancer (1986), 53, 345-353Br. J. Cancer (1986), 53, 345-353

WO2013/047639WO2013/047639

φ数百μｍ以上のオルガノイドを作製するためには、マトリクスの使用が必須であるが、オルガノイド周囲のマトリクスによって、オルガノイド同士の凝集・融合が阻害され、5 mm以上のサイズにオルガノイドを大型化することは困難である。また、マトリクスの使用は、GCTP準拠という観点から、再生医療等への応用を困難にする一つの要因となっている。 In order to produce organoids with a diameter of several hundred μm or more, it is essential to use a matrix. is difficult. In addition, the use of matrices is one of the factors that make application to regenerative medicine difficult from the viewpoint of GCTP compliance.

本発明は、マトリクスフリーで大型オルガノイドを作製する方法を提供することを目的とする。 An object of the present invention is to provide a matrix-free method for producing large organoids.

本発明者らは、培養容器中マトリクスフリーで臓器細胞と血管内皮細胞と間葉系細胞を共培養することでオルガノイド（細胞塊）を作製し、これらのオルガノイドを細胞接着可能な面上に播種し、オルガノイドを播種した面の表側及び裏側から培地を供給しながら培養することにより、任意の大きさの融合型オルガノイドを創出することに成功し、本発明を完成させるに至った。この融合型オルガノイドは内部に血管網構造を形成することを確認した。また、融合型オルガノイドにおいて、複数の臓器分化マーカーの遺伝子発現が上昇していた。本発明の方法で作製した融合型肝芽においては、肝機能関連遺伝子の発現レベルの上昇が認められ、毛細血管や細小動静脈、類洞、肝内胆管の形成も確認された。さらに、融合型オルガノイドをマウスに移植すると、マウスの血管系と融合型オルガノイドの血管系の吻合、血液灌流が確認された。 The present inventors prepared organoids (cell clusters) by co-cultivating organ cells, vascular endothelial cells, and mesenchymal cells in a culture vessel without matrix, and seeded these organoids on a surface capable of cell adhesion. Then, by culturing while supplying a medium from the front and back sides of the surface on which the organoids were seeded, we succeeded in creating fusion-type organoids of arbitrary size, and completed the present invention. It was confirmed that this fusion-type organoid forms a vascular network structure inside. In addition, gene expression of multiple organ differentiation markers was elevated in the fusion-type organoids. In the fused liver buds produced by the method of the present invention, an increase in the expression level of liver function-related genes was observed, and the formation of capillaries, arterioles and veins, sinusoids, and intrahepatic bile ducts was also confirmed. Furthermore, when the fusion-type organoids were transplanted into mice, anastomosis and blood perfusion between the vascular system of the mouse and the fusion-type organoids were confirmed.

本発明の要旨は以下の通りである。
（１）細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養することを含む、細胞塊融合法。
（２）播種面に対して細胞塊が占める面積の比率が４０～１００％となるように細胞塊を細胞接着可能な面上に播種する（１）記載の方法。
（３）細胞塊が血管細胞及び／又は間葉系細胞を含む（１）又は（２）に記載の方法。
（４）間葉系細胞と血管細胞の割合が１：０．０１～１００である（３）記載の方法。
（５）間葉系細胞と血管細胞の割合が１：０．１～１０である（４）記載の方法。
（６）融合された細胞塊が血管網構造を形成している（１）～（５）のいずれかに記載の方法。
（７）細胞塊が器官芽である（１）～（６）のいずれかに記載の方法。
（８）器官芽が、組織又は臓器細胞、間葉系細胞及び血管細胞から形成されたものである（７）記載の方法。
（９）組織又は臓器細胞、間葉系細胞及び血管細胞の割合が、１０：０．１～１０：０．１～１０である（８）記載の方法。
（１０）間葉系細胞と血管細胞の割合が１：０．１～１０である（９）記載の方法。
（１１）器官芽が細胞非接着面を有する培養器材中で形成されたものである（７）～（１０）のいずれかに記載の方法。
（１２）器官芽が肝芽であり、融合された肝芽が血管網構造を形成している（７）～（１１）のいずれかに記載の方法。
（１３）血管網構造が毛細血管及び／又は細小動静脈を含む（１２）記載の方法。
（１４）細胞塊が肝芽であり、融合された肝芽が胆管構造を形成している（６）～（１３）のいずれかに記載の方法。
（１５）細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養して、細胞塊を融合させることを含む、融合細胞塊の作製方法。
（１６）細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養し、血管網構造を形成することを含む、血管網構造を構築する方法。
（１７）肝細胞、間葉系細胞及び血管細胞から形成された肝芽をin vitroで融合することで作製され、血管網構造及び／又は胆管構造を有する人工臓器。
（１８）肝細胞、間葉系細胞及び血管細胞の少なくとも１つが、人工的に作製された多能性幹細胞から分化誘導されたものである（１７）記載の人工臓器。The gist of the present invention is as follows.
(1) A cell clump fusion method comprising seeding cell clumps on a cell-adherable surface and culturing while supplying a culture medium from the front and back sides of the surface on which the cell clumps are seeded.
(2) The method according to (1), in which the cell aggregates are sown on a cell-adherable surface such that the ratio of the area occupied by the cell aggregates to the seeding surface is 40 to 100%.
(3) The method according to (1) or (2), wherein the cell mass contains vascular cells and/or mesenchymal cells.
(4) The method according to (3), wherein the ratio of mesenchymal cells to vascular cells is 1:0.01-100.
(5) The method according to (4), wherein the ratio of mesenchymal cells to vascular cells is 1:0.1-10.
(6) The method according to any one of (1) to (5), wherein the fused cell mass forms a vascular network structure.
(7) The method according to any one of (1) to (6), wherein the cell mass is an organ bud.
(8) The method according to (7), wherein the organ buds are formed from tissue or organ cells, mesenchymal cells and vascular cells.
(9) The method according to (8), wherein the ratio of tissue or organ cells, mesenchymal cells and vascular cells is 10:0.1-10:0.1-10.
(10) The method according to (9), wherein the ratio of mesenchymal cells to vascular cells is 1:0.1-10.
(11) The method according to any one of (7) to (10), wherein the organ buds are formed in a cultureware having a cell non-adhesive surface.
(12) The method according to any one of (7) to (11), wherein the organ buds are liver buds, and the fused liver buds form a vascular network structure.
(13) The method according to (12), wherein the vascular network structure includes capillaries and/or arterioles and veins.
(14) The method according to any one of (6) to (13), wherein the cell mass is a liver bud, and the fused liver buds form a bile duct structure.
(15) Production of fused cell clusters, including seeding cell clusters on a surface capable of cell adhesion, culturing while supplying a medium from the front and back sides of the surface on which the cell clusters are seeded, and fusing the cell clusters. Method.
(16) Constructing a vascular network structure, including seeding cell clusters on a cell-adherable surface and culturing while supplying a culture medium from the front and back sides of the surface on which the cell clusters are seeded to form a vascular network structure. how to.
(17) An artificial organ produced by in vitro fusion of liver buds formed from hepatocytes, mesenchymal cells and vascular cells and having a vascular network structure and/or bile duct structure.
(18) The artificial organ according to (17), wherein at least one of hepatocytes, mesenchymal cells and vascular cells is induced to differentiate from artificially produced pluripotent stem cells.

本発明により、mmオーダー以上の融合細胞塊を作製することができる。また、本発明により、融合細胞塊に血管ネットワーク様の構造を再現良く形成することができる。さらに、本発明により、肝機能関連遺伝子の発現レベルが上昇した融合型肝芽を作製することができ、融合型肝芽では、毛細血管や細小動静脈、類洞、肝内胆管が形成されうる。
本発明により、血管網構造を有する融合オルガノイドが安定的に作製可能となるため、大血管等の組織と融合させることで将来的に移植可能な臓器が作製可能となる。According to the present invention, a fused cell mass of mm order or more can be produced. Moreover, according to the present invention, a vascular network-like structure can be formed in a fused cell cluster with good reproducibility. Furthermore, according to the present invention, fusion-type liver buds with increased expression levels of liver function-related genes can be produced, and capillaries, arterioles and veins, sinusoids, and intrahepatic bile ducts can be formed in the fusion-type liver buds. .
According to the present invention, it is possible to stably produce a fusion organoid having a vascular network structure, so that it becomes possible to produce an organ that can be transplanted in the future by fusing it with a tissue such as a large blood vessel.

本発明により、大型のオルガノイドを作製することが可能となった。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2018‐061095の明細書および／または図面に記載される内容を包含する。The present invention has made it possible to produce large organoids.
This specification includes the contents described in the specification and/or drawings of the Japanese patent application, Japanese Patent Application No. 2018-061095, which is the basis of the priority of this application.

小型肝芽を用いた細胞塊融合の検討結果を示す。The results of examination of cell mass fusion using small liver buds are shown. 従来法肝芽（マトリゲル上で作製された肝芽）を用いた細胞塊融合の検討結果を示す。Fig. 3 shows the results of examination of cell mass fusion using conventional liver buds (liver buds produced on Matrigel). 融合型肝芽における組織再構成の結果を示す。The results of tissue reconstruction in fused liver buds are shown. 小型肝芽と融合型肝芽の肝分化マーカー解析結果を示す。Fig. 2 shows the results of hepatic differentiation marker analysis of small hepatic buds and fused hepatic buds. 従来法肝芽と融合型肝芽の肝機能解析の結果を示す。The results of liver function analysis of conventional liver buds and fused liver buds are shown. 融合型肝芽のクラニアルウィンドウ移植の結果を示す。Results of cranial window implantation of fused liver buds are shown. 細胞構成比率の違いによる血管網形成の制御の結果を示す。The results of control of vascular network formation due to differences in cell composition ratio are shown. 融合型肝芽のサイズ制御の結果を示す。Results of size control of fused liver buds are shown. 細胞塊の大きさと播種密度の検討結果を示す。The results of examining the size of cell clusters and the seeding density are shown. 細胞比率検討の結果を示す。The result of cell ratio examination is shown.

以下、本発明を詳細に説明する。 The present invention will be described in detail below.

本発明は、細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養することを含む、細胞塊融合法を提供する。本発明の方法により、融合細胞塊が得られる。よって、本発明は、細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養して、細胞塊を融合させることを含む、融合細胞塊の作製方法を提供する。本発明の方法により、細胞塊に血管網構造を構築できる。よって、本発明は、細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養し、血管網構造を形成することを含む、血管網構造を構築する方法を提供する。
本発明の方法により、これまでのマトリクスフリーで細胞塊を作製する方法では報告のない大きさ、すなわち、φ8 mm以上の細胞塊を作製することができる。Br. J. Cancer (1986), 53, 345-353では、プロットしてあるデータからは、φ2.2 mm大くらいまでの細胞塊が形成されているようであり、その他の論文では、回転培養装置でφ1.1 mm大までの細胞塊が作製可能 (Zanoni M et al., Sci. Rep. 6, 19103. 2016)、Uボトムプレートでφ1 mm大の細胞塊を作製可能(Vinci M et al., BMC Biology 10:29, 2012)、Uボトムプレートで細胞塊を作製した後低吸着プレート上での旋回培養を行うことでφ4 mm大の細胞塊を作製可能(Xiang Y et al., Cell Stem Cell 21, 383-398, 2017)、セルカルチャーインサート上でφ4 mm大までの細胞塊が作製可能[Takasato M et al., Nature 526, 564-568, 2015]なようである。また、マトリクスを使用してオルガノイドを作製する方法では、最大で5mm程度の大きさが限界であるようだが（Chen Y et al., Nat. Cell Biol. 19, 542-549）、本発明の方法では、この報告よりも大きな細胞塊を作製できる。The present invention provides a cell mass fusion method comprising seeding cell masses on a cell-adherable surface and culturing while supplying culture media from the front and back sides of the surface on which the cell masses are seeded. A fusion cell mass is obtained by the method of the present invention. Therefore, the present invention involves seeding cell clusters on a cell-adherable surface, culturing while supplying a medium from the front and back sides of the surface on which the cell clusters are seeded, and fusing the cell clusters. A method of making a mass is provided. According to the method of the present invention, a vascular network structure can be constructed in a cell cluster. Therefore, the present invention provides a vascular network comprising seeding cell clusters on a surface capable of cell adhesion and culturing while supplying a medium from the front and back sides of the surface on which the cell clusters are seeded to form a vascular network structure. It provides a way to build structures.
According to the method of the present invention, it is possible to prepare cell aggregates having a size not reported by conventional matrix-free methods for preparing cell aggregates, that is, cell aggregates having a diameter of 8 mm or more. In Br. J. Cancer (1986), 53, 345-353, from the plotted data, it seems that cell clusters up to φ2.2 mm are formed. The apparatus can produce cell aggregates up to φ1.1 mm (Zanoni M et al., Sci. Rep. 6, 19103. 2016), and the U-bottom plate can produce cell aggregates up to φ1 mm (Vinci M et al. ., BMC Biology 10:29, 2012), φ4 mm large cell clusters can be prepared by spinning culture on a low-adsorption plate after preparing cell clusters on a U-bottom plate (Xiang Y et al., Cell Stem Cell 21, 383-398, 2017), and cell clusters up to φ4 mm in size can be produced on cell culture inserts [Takasato M et al., Nature 526, 564-568, 2015]. In addition, in the method of preparing organoids using a matrix, the maximum size seems to be about 5 mm (Chen Y et al., Nat. Cell Biol. 19, 542-549), but the method of the present invention can produce cell clusters larger than those reported here.

細胞塊は、器官芽（オルガノイド）、スフェロイドなど、いかなる細胞の集合体であってもよいが、血管細胞（例えば、血管内皮細胞）と間葉系細胞（例えば、間葉系幹細胞のような未分化間葉系細胞）のどちらか一方又は両方を含むものであるとよい。間葉系細胞と血管細胞の割合は１：０．０１～１００であるとよく、好ましくは、１：０．１～１０であり、より好ましくは、１：０．５～２である。細胞塊が血管細胞と間葉系細胞を含むと、融合した細胞塊に血管網構造が形成されうる。
スフェロイドは、細胞同士が集合・凝集化した球状の細胞集合体であり、三次元構造をとっている。
「器官芽」とは、成熟することで器官に分化することができる構造体であって、その一例として、WO2013/047639では、組織又は臓器細胞、血管細胞（例えば、血管内皮細胞）、及び間葉系細胞（例えば、間葉系幹細胞のような未分化間葉系細胞若しくはそれから分化した細胞）の三種類の細胞から器官芽を作製する方法が開示されており、本発明では、この方法で作製した器官芽を好適に用いることができる。ある構造体が器官芽であるかどうかは、例えば、その構造体を生体へ移植し、目的の器官に分化できるかどうかを調べること（目的の器官へ分化していれば器官芽であると判断できる。）、及び／又はその構造体が上述した三種類の細胞をすべて含んでいるかどうかを調べること（三種類の細胞をすべて含んでいれば器官芽であると判断できる。）により確認できる。器官芽は、例えば、腎臓、心臓、肺臓、脾臓、食道、胃、甲状腺、副甲状腺、胸腺、生殖腺、脳、脊髄などの器官に分化する器官芽などであってもよいが、肝臓に分化する器官芽（肝芽）、膵臓に分化する器官芽（膵芽）、腸管に分化する器官芽など内胚葉性器官に分化する器官芽が好ましい。ある構造体が内胚葉性器官に分化する器官芽であるかどうかは、マーカーとなるタンパク質の発現を調べることにより確認できる（後述するマーカータンパク質のいずれか一つあるいは複数が発現していれば器官芽であると判断できる。）。例えば、肝芽では、HHEX、SOX2、HNF4A、AFP、 ALBなどがマーカーになり、膵芽では、PDX1、SOX17、SOX9などがマーカーになり、腸管に分化する器官芽では、CDX2、SOX9などがマーカーになる。当業者間で使用されている用語のうち、liver bud、liver diverticula、liver organoid、pancreatic (dorsal or ventral) buds、pancreatic diverticula、pancreatic organoid、intestinal bud、intestinal diverticula、intestinal organoid（K. Matsumoto, et al. Science.19;294(5542):559-63.(2001)）などは本発明における器官芽に含まれる。The cell mass may be an aggregate of any cells such as organ buds (organoids), spheroids, etc., but may include vascular cells (e.g., vascular endothelial cells) and mesenchymal cells (e.g., mesenchymal stem cells). differentiated mesenchymal cells). The ratio of mesenchymal cells to vascular cells is preferably 1:0.01-100, preferably 1:0.1-10, more preferably 1:0.5-2. When the cell mass contains vascular cells and mesenchymal cells, a vascular network structure can be formed in the fused cell mass.
A spheroid is a spherical cell aggregate in which cells aggregate and agglomerate, and has a three-dimensional structure.
An "organ bud" is a structure that can be differentiated into an organ by maturation. Disclosed is a method for producing organ buds from three types of cells: mesenchymal cells (e.g., undifferentiated mesenchymal cells such as mesenchymal stem cells or cells differentiated therefrom). The produced organ buds can be preferably used. Whether or not a certain structure is an organ bud can be determined, for example, by transplanting the structure into a living body and examining whether it can differentiate into the target organ (if it has differentiated into the target organ, it is determined to be an organ bud). and/or by examining whether the structure contains all three types of cells (if all three types of cells are included, it can be determined to be an organ bud). An organ bud can be, for example, an organ bud that differentiates into an organ such as kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonad, brain, spinal cord, but differentiates into liver. Organ buds that differentiate into endodermal organs, such as organ buds (liver buds), organ buds that differentiate into the pancreas (pancreatic buds), and organ buds that differentiate into the intestinal tract, are preferred. Whether or not a given structure is an organ bud that differentiates into an endodermal organ can be confirmed by examining the expression of a marker protein (if any one or more of the marker proteins described later are expressed, the organ bud can be determined to be.). For example, HHEX, SOX2, HNF4A, AFP, and ALB are markers for liver buds, PDX1, SOX17, and SOX9 are markers for pancreatic buds, and CDX2 and SOX9 are markers for organ buds that differentiate into the intestinal tract. become. Among the terms used among those skilled in the art, liver bud, liver diverticula, liver organoid, pancreatic (dorsal or ventral) buds, pancreatic diverticula, pancreatic organoid, intestinal bud, intestinal diverticula, intestinal organoid (K. Matsumoto, et al. Science.19;294(5542):559-63.(2001)) are included in the organ buds in the present invention.

本発明において「組織又は臓器細胞」とは、組織又は臓器を構成する機能細胞、又は機能細胞へと分化する未分化細胞をいう。「未分化な組織又は臓器細胞」としては、例えば、腎臓、心臓、肺臓、脾臓、食道、胃、甲状腺、副甲状腺、胸腺、生殖腺、脳、脊髄などの器官に分化可能な細胞などであってもよく、脳、脊髄、副腎髄質、表皮、毛髪・爪・皮膚腺、感覚器、末梢神経、水晶体などの外胚葉性器官に分化可能な細胞、腎臓、尿管、心臓、血液、生殖腺、副腎皮質、筋肉、骨格、真皮、結合組織、中皮などの中胚葉性器官に分化可能な細胞、肝臓、膵臓、腸管、肺、甲状腺、副甲状腺、尿路などの内胚葉性器官に分化可能な細胞などを挙げることができる。ある細胞が外胚葉性器官、中胚葉性器官又は内胚葉性器官に分化可能な細胞であるかどうかは、マーカーとなるタンパク質の発現を調べることにより確認できる（マーカータンパク質のいずれか一つあるいは複数が発現していれば内胚葉性器官に分化可能な細胞であると判断できる。）。例えば、肝臓に分化可能な細胞では、HHEX、SOX2、HNF4A、AFP、 ALBなどがマーカーになり、膵臓に分化可能な細胞では、PDX1、SOX17、SOX9などがマーカーになり、腸管に分化可能な細胞では、CDX2、SOX9などがマーカーになり、腎臓に分化可能な細胞では、SIX2、SALL1、心臓に分化可能な細胞では、NKX2-5 MYH6、ACTN2、MYL7、HPPA、血液に分化可能な細胞では、C-KIT、SCA1、TER119、HOXB4、脳や脊髄に分化可能な細胞では、HNK1、AP2、NESTINなどがマーカーになる。当業者間で使用されている用語のうち、hepatoblast、hepatic progenitor cells、hepatic precursor cells、pancreatoblast、pancreatic progenitors、pancreatic progenitor cells、pancreatic precursor cells、endocrine precursors、intestinal progenitor cells、intestinal precursor cells、intermediate mesoderm、metanephric mesenchymal precursor cells、multipotent nephron progenitor、renal progenitor cell、cardiac mesoderm、cardiovascular progenitor cells、cardiac progenitor cells、（JR. Spence, et al. Nature.;470(7332):105-9.(2011)、Self, et al. EMBO J.; 25(21): 5214-5228.(2006)、J. Zhang, et al. Circulation Research.; 104: e30-e41(2009)、G. Lee, et al. Nature Biotechnology 25, 1468 - 1475 (2007)）などは本発明における未分化な組織又は臓器細胞に含まれる。未分化な組織又は臓器細胞は、組織又は臓器から採取したり、あるいは、人工多能性幹細胞（iPS細胞）、胚性幹細胞（ES細胞）などの多能性幹細胞から公知の方法に従って作製することができる。また、未分化な組織又は臓器細胞は、iPS細胞などの多能性幹細胞から組織又は臓器への分化が中途段階にある細胞、例えば、原始腸内胚葉細胞（Primitive Gut Endoderm Cells: PGECs）（特許第5777127号）などであってもよい。PGECsは、肝細胞、膵細胞及び腸細胞への分化が可能であり（分化機能が高い）、癌の悪性度に関係するマーカーを発現せず（安全性が高い）、iPS細胞から非フィーダー環境下で分化誘導して調製することができるので、臨床応用も可能とするという利点を持つ。しかも、PGECsは、大量調製が可能である。PGECsは、特許第5777127号に記載の方法で調製することができる。あるいは、無血清培地にアクチビンを添加してiPS細胞などの多能性幹細胞を培養することで、CXCR4及びE-cadherin両陽性の内胚葉系細胞に誘導したものや、さらに、得られた内胚葉系細胞をBMP４、FGF２を添加して２日間培養することで得られる、CXCR4陰性 HNF4α陽性の肝臓内胚葉集団であってもよい。その他、例えば、肝臓に分化可能な臓器細胞は、K.Si-Taiyeb, et al. Hepatology, 51 (1): 297- 305(2010)、T. Touboul, et al. Hepatology. 51(5):1754-65.(2010)に従って作製することができ、膵臓に分化可能な臓器細胞は、D. Zhang, et al. Cell Res.;19(4):429-38.(2009)に従って作製することができ、腸管に分化可能な臓器細胞は、J. Cai, et al. J Mol Cell Biol.;2(1):50-60(2010)、R. Spence, et al. Nature.;470(7332):105-9.(2011)に従って作製することができ、心臓に分化可能な臓器細胞は、J. Zhang, et al. Circulation Research.; 104: e30-e41(2009) に従って作製することができ、脳や脊髄に分化可能な細胞では、G. Lee, et al. Nature Biotechnology 25, 1468 - 1475 (2007) に従って作製することができる。「分化した組織又は臓器細胞」としては、膵臓の内分泌細胞、膵臓の膵管上皮細胞、肝臓の肝細胞、腸管の上皮細胞、腎臓の尿細管上皮細胞、腎臓の糸球体上皮細胞、心臓の心筋細胞、血液のリンパ球や顆粒球、赤血球、脳の神経細胞やグリア細胞、脊髄の神経細胞やシュワン細胞などを例示できる。組織又は臓器細胞は、がん化した組織や臓器に由来する細胞（がん細胞）であってもよい。がん細胞は、樹立がん細胞、がん患者（非ヒト動物も含む）に由来する初代培養がん細胞や継代培養がん細胞のいずれであってもよい。組織又は臓器細胞は、主としてヒト由来のものを用いるが、ヒト以外の動物（例えば、実験動物、愛玩動物、使役動物、競走馬、闘犬などに利用される動物、具体的には、マウス、ラット、ウサギ、ブタ、イヌ、サル、ウシ、ウマ、ヒツジ、ニワトリ、サメ、エイ、ギンザメ、サケ、エビ、カニなど）由来の組織又は臓器細胞を用いてもよい。 In the present invention, "tissue or organ cells" refer to functional cells that constitute tissues or organs, or undifferentiated cells that differentiate into functional cells. Examples of "undifferentiated tissue or organ cells" include cells capable of differentiating into organs such as kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonad, brain, and spinal cord. Cells capable of differentiating into ectodermal organs such as brain, spinal cord, adrenal medulla, epidermis, hair/nail/dermal gland, sensory organs, peripheral nerves, lens, kidney, ureter, heart, blood, gonad, adrenal cortex , cells capable of differentiating into mesodermal organs such as muscle, skeleton, dermis, connective tissue, and mesothelial cells; can be mentioned. Whether a given cell is a cell capable of differentiating into an ectodermal organ, a mesoderm organ, or an endodermal organ can be confirmed by examining the expression of a marker protein (any one or more of the marker proteins are expressed). If it is, it can be judged that the cell is capable of differentiating into an endodermal organ.). For example, HHEX, SOX2, HNF4A, AFP, and ALB are markers for cells that can differentiate into the liver, and PDX1, SOX17, and SOX9 are markers for cells that can differentiate into the pancreas. In , CDX2, SOX9, etc. are markers, SIX2, SALL1 in cells that can differentiate into kidney, NKX2-5 MYH6, ACTN2, MYL7, HPPA in cells that can differentiate into heart, and MYH6, ACTN2, MYL7, HPPA in cells that can differentiate into blood. C-KIT, SCA1, TER119, HOXB4, and HNK1, AP2, NESTIN, etc. are markers for cells that can differentiate into the brain and spinal cord. Among the terms used among those skilled in the art, hepatoblast, hepatic progenitor cells, hepatic precursor cells, pancreatoblast, pancreatic progenitors, pancreatic progenitor cells, pancreatic precursor cells, endocrine precursors, intestinal progenitor cells, intestinal precursor cells, intermediate mesoderm, metanephric mesenchymal precursor cells, multipotent nephron progenitor, renal progenitor cell, cardiovascular mesoderm, cardiovascular progenitor cells, cardiovascular progenitor cells, (JR. Spence, et al. Nature; 470(7332): 105-9. (2011), Self, et al. al. EMBO J.; 25(21): 5214-5228.(2006), J. Zhang, et al. Circulation Research.; 104: e30-e41(2009), G. Lee, et al. 1468-1475 (2007)) are included in the undifferentiated tissue or organ cells of the present invention. Undifferentiated tissue or organ cells are collected from tissues or organs, or prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. can be done. In addition, undifferentiated tissue or organ cells are cells that are in the middle stage of differentiation from pluripotent stem cells such as iPS cells into tissues or organs, such as primitive gut endoderm cells (PGECs) (patent No. 5777127) and the like. PGECs can differentiate into hepatocytes, pancreatic cells and enterocytes (high differentiation function), do not express markers related to cancer malignancy (high safety), and can be transferred from iPS cells to a non-feeder environment. Since it can be prepared by inducing differentiation under conditions, it has the advantage of enabling clinical application. Moreover, PGECs can be prepared in large quantities. PGECs can be prepared by the method described in Japanese Patent No. 5777127. Alternatively, by adding activin to a serum-free medium and culturing pluripotent stem cells such as iPS cells, CXCR4 and E-cadherin both positive endoderm cells were induced, or the obtained endoderm It may be a CXCR4-negative HNF4α-positive liver endoderm population obtained by adding BMP4 and FGF2 to the lineage cells and culturing them for 2 days. In addition, for example, organ cells capable of differentiating into liver are described in K. Si-Taiyeb, et al. Hepatology, 51 (1): 297-305 (2010), T. Touboul, et al. 1754-65. (2010), and organ cells capable of differentiating into the pancreas should be generated according to D. Zhang, et al. Cell Res.;19(4):429-38.(2009). J. Cai, et al. J Mol Cell Biol.;2(1):50-60 (2010), R. Spence, et al. Nature.;470(7332). ): 105-9. (2011), and organ cells capable of differentiating into heart can be generated according to J. Zhang, et al. Circulation Research.; 104: e30-e41 (2009). , cells capable of differentiating into brain and spinal cord can be prepared according to G. Lee, et al. Nature Biotechnology 25, 1468-1475 (2007). "Differentiated tissue or organ cells" include pancreatic endocrine cells, pancreatic ductal epithelial cells of pancreas, liver hepatocytes, intestinal epithelial cells, renal tubular epithelial cells, renal glomerular epithelial cells, and cardiac myocardial cells. , blood lymphocytes, granulocytes, red blood cells, brain nerve cells and glial cells, spinal cord nerve cells and Schwann cells. Tissue or organ cells may be cells (cancer cells) derived from cancerous tissues or organs. Cancer cells may be established cancer cells, primary cultured cancer cells derived from cancer patients (including non-human animals), or subcultured cancer cells. Tissues or organ cells are mainly derived from humans, but non-human animals (e.g., experimental animals, pets, working animals, racehorses, animals used for fighting dogs, specifically mice, rats, etc.) , rabbit, pig, dog, monkey, cow, horse, sheep, chicken, shark, ray, chimaera, salmon, shrimp, crab, etc.) may be used.

血管細胞は、臍帯静脈などの血管組織から分離することができるが、血管組織から分離された細胞に限定されることはなく、ｉＰＳ細胞やＥＳ細胞などの全能性あるいは多能性を有する細胞から分化誘導されたものであってもよい。血管細胞としては、血管内皮細胞、血管平滑筋細胞などを例示することができるが、血管内皮細胞が好ましく、臍帯静脈由来血管内皮細胞が市販されており、入手しやすい。本発明において「血管内皮細胞」とは、血管内皮を構成する細胞、又はそのような細胞に分化することのできる細胞をいう。ある細胞が血管内皮細胞であるかどうかは、マーカータンパク質、例えば、TIE2、VEGFR-1、VEGFR-2、VEGFR-3、CD41が発現しているかどうかを調べることにより確認できる（前記マーカータンパク質のいずれか一つあるいは複数が発現していれば血管内皮細胞であると判断できる。）。本発明において用いる血管内皮細胞は、分化したものであっても、未分化なものであってもよい。血管内皮細胞が、分化した細胞であるかどうかは、CD31、CD144により、確認することができる。当業者間で使用されている用語のうち、endothelial cells、umbilical vein endothelial cells、endothelial progenitor cells、endothelial precursor cells、vasculogenic progenitors、hemangioblast（HJ. joo, et al. Blood. 25;118(8):2094-104.(2011)）などは本発明における血管内皮細胞に含まれる。好ましい血管内皮細胞は、臍帯静脈由来の血管内皮細胞である。血管内皮細胞は、血管から採取したり、あるいは、人工多能性幹細胞（iPS細胞）、胚性幹細胞（ES細胞）などの多能性幹細胞から公知の方法に従って作製することができる。血管内皮細胞は、主としてヒト由来のものを用いるが、ヒト以外の動物（例えば、実験動物、愛玩動物、使役動物、競走馬、闘犬などに利用される動物、具体的には、マウス、ラット、ウサギ、ブタ、イヌ、サル、ウシ、ウマ、ヒツジ、ニワトリ、サメ、エイ、ギンザメ、サケ、エビ、カニなど）由来の血管内皮細胞を用いてもよい。 Vascular cells can be isolated from vascular tissues such as umbilical veins, but are not limited to cells isolated from vascular tissues, and include totipotent or pluripotent cells such as iPS cells and ES cells. Differentiation may be induced. Examples of vascular cells include vascular endothelial cells and vascular smooth muscle cells. Vascular endothelial cells are preferred, and umbilical vein-derived vascular endothelial cells are commercially available and readily available. In the present invention, "vascular endothelial cells" refer to cells that constitute vascular endothelium, or cells that can differentiate into such cells. Whether a certain cell is a vascular endothelial cell can be confirmed by examining whether marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD41 are expressed (any of the marker proteins If one or more of them are expressed, it can be determined that they are vascular endothelial cells.). Vascular endothelial cells used in the present invention may be differentiated or undifferentiated. Whether or not vascular endothelial cells are differentiated cells can be confirmed by CD31 and CD144. endothelial cells, umbilical vein endothelial cells, endothelial progenitor cells, endothelial precursor cells, vasculogenic progenitors, hemangioblast (HJ. joo, et al. Blood. 25;118(8):2094) -104.(2011)) are included in the vascular endothelial cells of the present invention. Preferred vascular endothelial cells are umbilical vein-derived vascular endothelial cells. Vascular endothelial cells can be collected from blood vessels or prepared from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) according to known methods. Vascular endothelial cells are mainly derived from humans, but animals other than humans (e.g., experimental animals, pets, working animals, race horses, animals used for fighting dogs, specifically mice, rats, etc.) Vascular endothelial cells derived from rabbit, pig, dog, monkey, cow, horse, sheep, chicken, shark, ray, chimaera, salmon, shrimp, crab, etc.) may also be used.

本発明において「間葉系細胞」とは、主として中胚葉に由来する結合織に存在し、組織で機能する細胞の支持構造を形成する結合織細胞であるが、間葉系細胞への分化運命が決定しているが、まだ間葉系細胞へ分化していない細胞も含む概念である。本発明において用いる間葉系細胞は、分化したものであっても、未分化なものであってもよい。ある細胞が未分化間葉系細胞であるかどうかは、マーカータンパク質、例えば、Stro-1、CD29、CD44、CD73、CD90、CD105、CD133、CD271、Nestinが発現しているかどうかを調べることにより確認できる（前記マーカータンパク質のいずれか一つあるいは複数が発現していれば未分化間葉系細胞であると判断できる。）。また、前項のマーカーのいずれも発現していない間葉系細胞は分化間葉系細胞と判断できる。当業者間で使用されている用語のうち、mesenchymal stem cells、mesenchymal progenitor cells、mesenchymal cells（R. Peters, et al. PLoS One. 30;5(12):e15689.(2010)）などは本発明における間葉系細胞に含まれる。好ましい間葉系細胞は、骨髄由来の間葉系細胞（特に、間葉系幹細胞）である。間葉系細胞は、骨髄、脂肪組織、胎盤組織、臍帯組織、歯髄等の組織から採取したり、あるいは、人工多能性幹細胞（iPS細胞）、胚性幹細胞（ES細胞）などの多能性幹細胞から公知の方法に従って作製することができる。間葉系細胞は、主としてヒト由来のものを用いるが、ヒト以外の動物（例えば、実験動物、愛玩動物、使役動物、競走馬、闘犬などに利用される動物、具体的には、マウス、ラット、ウサギ、ブタ、イヌ、サル、ウシ、ウマ、ヒツジ、ニワトリ、サメ、エイ、ギンザメ、サケ、エビ、カニなど）由来の未分化間葉系細胞を用いてもよい。 In the present invention, the term "mesenchymal cells" refers to connective tissue cells that exist mainly in connective tissue derived from mesoderm and form a support structure for cells that function in tissues. is determined, but the concept also includes cells that have not yet differentiated into mesenchymal cells. The mesenchymal cells used in the present invention may be differentiated or undifferentiated. Whether a certain cell is an undifferentiated mesenchymal cell is confirmed by examining whether marker proteins such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin are expressed. (If one or more of the above marker proteins are expressed, it can be determined that the cells are undifferentiated mesenchymal cells.). Mesenchymal cells that do not express any of the markers described above can be determined to be differentiated mesenchymal cells. Among the terms used by those skilled in the art, mesenchymal stem cells, mesenchymal progenitor cells, mesenchymal cells (R. Peters, et al. PLoS One. 30;5(12):e15689.(2010)) contained in mesenchymal cells in Preferred mesenchymal cells are bone marrow-derived mesenchymal cells (especially mesenchymal stem cells). Mesenchymal cells are collected from tissues such as bone marrow, adipose tissue, placental tissue, umbilical cord tissue, and dental pulp, or pluripotent cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). It can be produced from stem cells according to a known method. Mesenchymal cells mainly derived from humans are used, but non-human animals (e.g., experimental animals, pets, working animals, racehorses, animals used for fighting dogs, specifically mice, rats, etc.) , rabbit, pig, dog, monkey, cow, horse, sheep, chicken, shark, ray, chimaera, salmon, shrimp, crab, etc.) may be used.

組織又は臓器細胞、血管内皮細胞及び間葉系細胞の３種類の細胞全てをiPS細胞から作製し、これらの細胞から器官芽を創製する方法は、Takebe T et al., 2017, Cell Reports 21, 2661-2670に記載されており、これらの細胞や器官芽を本発明に用いてもよい。 Takebe T et al., 2017, Cell Reports 21, 2017, Cell Reports 21, 2661-2670, and these cells and organ buds may be used in the present invention.

共培養における三種類の細胞の培養比は器官芽が形成できる範囲内であれば特に限定されないが、好適な細胞の数比は、組織又は臓器細胞：血管細胞：間葉系細胞＝１０：０．１～１０：０．１～１０である。血管細胞と間葉系細胞が少なくなると、血管の形成が困難になる。間葉系細胞が多くなると、細胞接着可能な面への接着よりも、細胞同士の接着力が強まることにより収縮してしまうことが多くなり、細胞塊の融合が難しくなる。血管細胞が多くなると、血管構築に必要な間葉系細胞の量が足りなくなることで血管形成が阻害される。
間葉系細胞と血管細胞の割合（数比）は１：０．０１～１００であるとよく、好ましくは、１：０．１～１０であり、より好ましくは、１：０．５～２である。組織又は臓器細胞23万個程度、血管内皮細胞16万個程度、間葉系細胞16万個程度を共培養して、大きさが３，５００～４，５００マイクロメートル程度の器官芽を形成させることができる。間葉系細胞の比率が高くなるほど、器官芽に形成される血管網の網目構造が細かくなる。血管網構造を形成するのに好適な細胞の数比は、組織又は臓器細胞：血管内皮細胞：間葉系細胞＝１０：１～１０：１～１０、より好ましくは１０：２～７：２～７である。
器官芽の形成のために、細胞を共培養する際に使用する培地は、器官芽が形成されるものであればどのようなものでもよいが、血管内皮細胞培養用の培地、組織又は臓器細胞培養用の培地、前記２つの培地を混合したものなどを使用することが好ましい。血管内皮細胞培養用の培地はどのようなものを使用してもよいが、hEGF（組換えヒト上皮細胞成長因子）、VEGF（血管内皮細胞成長因子）、ヒドロコルチゾン、bFGF、アスコルビン酸、IGF1、FBS、Antibiotics(例えば、ゲンタマイシン、アンフォテリシンBなど)、Heparin、L-Glutamine、Phenolred、BBEの少なくとも１種を含むものを使用するのが好ましい。血管内皮細胞培養用の培地としては、EGM-2 BulletKit（Lonza社製）、EGM BulletKit（Lonza社製）、VascuLife EnGS Comp Kit（LCT社製）、Human Endothelial-SFM Basal Growth Medium（Invitrogen社製）、ヒト微小血管内皮細胞増殖培地（TOYOBO社製）などを用いることができる。組織又は臓器細胞培養用の培地はどのようなものを使用してもよいが、臓器細胞が肝細胞である場合、ascorbic acid、BSA- FAF、insulin、hydrocortisone、GA-1000の少なくとも１種を含むものを使用するのが好ましい。肝細胞培養用の培地としては、HCM BulletKit（Lonza社製）よりhEGF（組換えヒト上皮細胞成長因子）を除いたもの、RPMI1640（Sigma-Aldrich社製）に1% B27 Supplements (GIBCO社製) と 10ng/mL hHGF (Sigma-Aldrich社製)などを用いることができる。ヒト肝芽の形成に関しては、EGM BulletKit（Lonza社製）とHCM BulletKit（Lonza社製）よりhEGF（組換えヒト上皮細胞成長因子）を除いたものを１：１で混ぜたものに、Dexamethasone、Oncostatin M、HGFを添加すると、肝芽の成熟に効果があることが判明している。
器官芽の形成のための細胞の培養温度は特に限定されないが、３０～４０℃とするのが好ましく、３７℃とするのが更に好ましい。
器官芽の形成のための細胞の培養期間は特に限定されないが、３～１４日とするのが好ましく、１０日とするのが更に好ましい。The culture ratio of the three types of cells in the co-culture is not particularly limited as long as it is within the range where organ buds can be formed. .1-10: 0.1-10. With fewer vascular cells and mesenchymal cells, blood vessel formation becomes more difficult. When the number of mesenchymal cells increases, the adhesion between the cells is strengthened rather than the adhesion to the cell-adhesive surface, causing contraction in many cases, making it difficult to fuse the cell clusters. When the number of vascular cells increases, the amount of mesenchymal cells required for angiogenesis becomes insufficient, which inhibits angiogenesis.
The ratio (number ratio) of mesenchymal cells and vascular cells is preferably 1:0.01 to 100, preferably 1:0.1 to 10, more preferably 1:0.5 to 2. is. About 230,000 tissue or organ cells, about 160,000 vascular endothelial cells, and about 160,000 mesenchymal cells are co-cultured to form organ buds with a size of about 3,500 to 4,500 micrometers. be able to. The higher the proportion of mesenchymal cells, the finer the mesh structure of the vascular network formed in the organ buds. The number ratio of cells suitable for forming a vascular network structure is tissue or organ cells: vascular endothelial cells: mesenchymal cells = 10: 1 to 10: 1 to 10, more preferably 10: 2 to 7: 2. ~7.
For the formation of organ buds, the medium used in co-culturing the cells may be any medium in which organ buds are formed. It is preferable to use a culture medium, a mixture of the above two media, or the like. Any medium may be used for culturing vascular endothelial cells, but hEGF (recombinant human epidermal growth factor), VEGF (vascular endothelial growth factor), hydrocortisone, bFGF, ascorbic acid, IGF1, FBS , Antibiotics (eg, gentamicin, amphotericin B, etc.), Heparin, L-Glutamine, Phenolred, BBE. As media for vascular endothelial cell culture, EGM-2 Bullet Kit (manufactured by Lonza), EGM Bullet Kit (manufactured by Lonza), VascuLife EnGS Comp Kit (manufactured by LCT), Human Endothelial-SFM Basal Growth Medium (manufactured by Invitrogen) , human microvascular endothelial cell growth medium (manufactured by TOYOBO) and the like can be used. Any medium may be used for tissue or organ cell culture, but when the organ cells are hepatocytes, it contains at least one of ascorbic acid, BSA-FAF, insulin, hydrocortisone, and GA-1000. It is preferable to use the The medium for hepatocyte culture is HCM Bullet Kit (manufactured by Lonza) without hEGF (recombinant human epidermal growth factor), RPMI1640 (manufactured by Sigma-Aldrich) and 1% B27 Supplements (manufactured by GIBCO). and 10 ng/mL hHGF (manufactured by Sigma-Aldrich) and the like can be used. For the formation of human liver buds, a 1:1 mixture of EGM Bullet Kit (manufactured by Lonza) and HCM Bullet Kit (manufactured by Lonza) from which hEGF (recombinant human epidermal growth factor) was removed was mixed with dexamethasone, Addition of Oncostatin M and HGF was found to be effective in the maturation of liver buds.
The temperature for culturing cells for forming organ buds is not particularly limited, but is preferably 30 to 40°C, more preferably 37°C.
The period of culturing cells for forming organ buds is not particularly limited, but is preferably 3 to 14 days, more preferably 10 days.

本発明において、細胞塊（例えば、器官芽）は、マトリクスのような細胞接着可能な面を用いずに（マトリクスフリー）作製されたものであるとよく、例えば、細胞非接着面を有する培養器材中で形成されたものであるとよい。
細胞非接着面を有する培養器材は、低吸着表面処理されたものであるとよく、例えば、細胞非接着性のポリマーが培養面にコートされているものであるとよい。細胞非接着性のポリマーとしては、リン脂質、リン脂質・高分子複合体、ポリ（２－ヒドロキシエチルメタクリレート）（ＰＨＥＭＡ）、ポリビニルアルコール、アガロース、キトサン、ポリエチレングリコール、アルブミン、光架橋超親水性ポリマーなどを例示することができるが、これらに限定されるわけではない。
また、培養器材の底部は、半球形状又は円錐台形状の凹みを多数有するものであるとよい。例えば、半球形状又は円錐台形状の凹み（凹みの容積は0.068 mm3）を１ウェルに６００個有する２４ウェルプレートの培養器材を用いて、１ウェル当たり総数20万～300万個の細胞を培養して、細胞塊を形成させるとよい。この時、細胞塊の大きさは、80～500マイクロメートルとなりうる。上記の培養器材及び培養条件はWO2015/182159に記載されており、本明細書において参考として引用する。細胞非接着面を有する培養器材としては、Elplasia RB 500 400 NA（クラレ）、96ウェルUボトムプレート又はVボトムプレート（住友ベークライト）などがあり、本発明において、好適に使用することができる。In the present invention, the cell mass (eg, organ bud) is preferably prepared without using a cell-adhesive surface such as a matrix (matrix-free). It may be formed in the
The cultureware having a cell-non-adhesive surface is preferably treated with a low-adsorption surface, for example, the culture surface is coated with a cell-non-adhesive polymer. Cell non-adhesive polymers include phospholipids, phospholipid/polymer complexes, poly(2-hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, and photocrosslinked superhydrophilic polymers. etc. can be exemplified, but are not limited to these.
In addition, the bottom of the cultureware preferably has a large number of hemispherical or truncated conical depressions. For example, a total of 200,000 to 3,000,000 cells are cultured per well using a 24-well plate cultureware having 600 hemispherical or truncated conical depressions (the volume of the depressions is 0.068 mm 3 ) per well. to form cell clusters. At this time, the size of the cell cluster can be 80-500 micrometers. The cultureware and culture conditions described above are described in WO2015/182159, which is incorporated herein by reference. Examples of cultureware having a cell-non-adhesive surface include Elplasia RB 500 400 NA (Kuraray), 96-well U-bottom plate, V-bottom plate (Sumitomo Bakelite), and the like, which can be preferably used in the present invention.

本発明の方法において、播種面に対して細胞塊が占める面積の比率が４０～１００％となるように細胞塊を細胞接着可能な面上に播種するとよく、播種面に対して細胞塊が占める面積の比率は、６０～１００％が好ましく、８０～１００％がより好ましい。播種面に対して細胞塊が占める面積の比率は、細胞塊の投射陰影面積を測定し、播種面の面積に対する比率を算出することにより得られる。細胞塊の投射陰影面積は以下の方法により測定することができる。FIJI, ImageJ, Photoshopなどの画像解析ソフトにより細胞塊の投射陰影面積を算出する。
本発明の方法において、細胞塊は高密度で細胞接着可能な面上に播種されうる。高密度とは、例えば、直径150 umの細胞塊の場合に空間1 cm3当たりに存在する細胞塊の数が、9.5 x 104 ～3.8 x 105個であるとよく、好ましくは、1.9 x 105～3.8 x 105個であり、より好ましくは、2.9 x 105～3.8 x 105個である。
細胞塊の数は、2個以上であるとよく、細胞塊の数を増やせば、より大きな融合細胞塊を作製することができる（後述の実施例参照）。
細胞塊の大きさは、80～500μｍが適当であり、100～250μｍが好ましい。
培地は、細胞塊の培養に適したものであればよく、例えば、細胞塊が肝芽である場合、EGM BulletKit（Lonza社製）とHCM BulletKit（Lonza社製）よりhEGF（組換えヒト上皮細胞成長因子）を除いたものを１：１で混ぜたものに、Dexamethasone、Oncostatin M、HGFを添加した培地、EGM BulletKit（Lonza社製）とVascuLife EnGS Comp Kit（LCT社製）を１：１で混合した培地、EGM BulletKit（Lonza社製）とEndothelial Cell Growth Medium MV（社製）を１：１で混合した培地などの培地が好ましいが、これらに限定されるわけではない。
「細胞塊同士の融合」とは、複数の細胞塊が連続した構造体を形成することをいい、融合した細胞塊は、内部が自己組織化して連結した血管構造を構築しうる。細胞塊同士が融合することで、細胞塊が大型化されるだけでなく、細胞塊に血管網構造が形成されるようになったり、血管網構造がさらに発達したり、また、細胞塊の機能が向上しうる。In the method of the present invention, the cell clusters are preferably seeded on the cell-adherable surface so that the ratio of the area occupied by the cell clusters to the seeding surface is 40 to 100%, and the cell clusters occupy the seeding surface. The area ratio is preferably 60 to 100%, more preferably 80 to 100%. The ratio of the area occupied by the cell cluster to the seeding surface can be obtained by measuring the projected shadow area of the cell cluster and calculating the ratio to the area of the seeding surface. The projected shadow area of the cell mass can be measured by the following method. Calculate the projected shadow area of the cell mass using image analysis software such as FIJI, ImageJ, and Photoshop.
In the method of the present invention, cell clusters can be seeded onto a surface that is dense and capable of cell attachment. High density means, for example, in the case of cell clusters with a diameter of 150 μm, the number of cell clusters present per 1 cm 3 of space is 9.5×10 4 to 3.8×10 5 , preferably 1.9×10 5 . 10 5 to 3.8 x 10 5 , more preferably 2.9 x 10 5 to 3.8 x 10 5 .
The number of cell clusters is preferably 2 or more, and by increasing the number of cell clusters, larger fused cell clusters can be produced (see Examples below).
The size of the cell mass is suitably 80-500 μm, preferably 100-250 μm.
Any medium may be used as long as it is suitable for culturing cell aggregates. For example, when the cell aggregates are liver buds, hEGF (recombinant human epithelial cell growth factors) were mixed 1:1, medium supplemented with dexamethasone, Oncostatin M, HGF, EGM Bullet Kit (manufactured by Lonza) and VascuLife EnGS Comp Kit (manufactured by LCT) at 1:1. A medium such as a mixed medium or a 1:1 mixture of EGM Bullet Kit (manufactured by Lonza) and Endothelial Cell Growth Medium MV (manufactured by Co.) is preferred, but not limited to these.
“Fusion between cell clusters” refers to formation of a continuous structure by a plurality of cell clusters, and the fused cell clusters can construct a vascular structure in which the insides are self-organized and connected. The fusion of cell clusters not only enlarges the cell clusters, but also forms a vascular network structure in the cell clusters, further develops the vascular network structure, and improves the function of the cell clusters. can improve.

本発明において、細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養することで、細胞塊が融合される。
細胞塊の融合は、細胞接着可能な面上で行われる。例えば、細胞接着可能な面が多孔性メンブレンの構造をとっていると、融合型の細胞塊を上下から栄養供給可能な点や酸素供給の面で融合後の培養に有利であると考えられるが、この態様に限定されるわけではない。細胞接着可能な面としては、大気下コロナ放電や真空ガスプラズマ重合処理（細胞接着表面処理）などにより、マイナスにチャージし、親水性を持つようになったもの、表面がゼラチン処理されたもの、細胞外マトリクス（コラーゲン、ラミニン、フィブロネクチンなど）やムコ多糖（ヘパリン硫酸、ヒアルロン酸、コンドロイチン硫酸など）でコーティングしたもの、塩基性合成ポリマー（ポリ-D-リシンなど）でコーティングしたもの、合成ナノファイバー表面を持つもの、親水性で中性なハイドロゲル層の表面を持つもの、コラーゲン膜(高研)などを例示することができるが、これらに限定されるわけではない。細胞接着可能な面が多孔性メンブレンの構造をとっている場合、ポアサイズは、0.4～8 μmであるとよい。細胞接着可能な面を有する培養器材としては、Falcon セルカルチャープレート（Corning）、Falconマルチセルカルチャープレート（Corning）、Falconセルカルチャーインサート（Corning）などがあり、本発明において、好適に使用することができる。培養は、回分培養、半回分培養（流加培養）、連続培養（灌流培養）のいずれの方法でもよい。また、静置培養、通気培養、攪拌培養、振盪培養、回転培養のいずれであってもよいが、静置培養が好ましい。In the present invention, the cell clusters are fused by seeding the cell clusters on a cell-adherable surface and culturing while supplying medium from the front and back sides of the surface on which the cell clusters are seeded.
Fusion of cell clusters is performed on surfaces that allow cell adhesion. For example, if the cell-adherable surface has a porous membrane structure, it is considered to be advantageous for post-fusion culture in terms of nutrient supply and oxygen supply to the fused cell mass from above and below. , but not limited to this embodiment. Surfaces that can adhere to cells include surfaces that have been negatively charged and made hydrophilic by atmospheric corona discharge or vacuum gas plasma polymerization treatment (cell adhesion surface treatment), surfaces that have been gelatinized, Coated with extracellular matrices (collagen, laminin, fibronectin, etc.) or mucopolysaccharides (heparin sulfate, hyaluronic acid, chondroitin sulfate, etc.), coated with basic synthetic polymers (poly-D-lysine, etc.), synthetic nanofibers Examples include, but are not limited to, those having a surface, those having a hydrophilic and neutral hydrogel layer surface, collagen membranes (Koken), and the like. When the cell-adherable surface has a porous membrane structure, the pore size is preferably 0.4 to 8 μm. Examples of cultureware having a cell-adherable surface include Falcon cell culture plate (Corning), Falcon multi-cell culture plate (Corning), Falcon cell culture insert (Corning), etc., and can be preferably used in the present invention. . The culture may be any of batch culture, semi-batch culture (fed-batch culture), and continuous culture (perfusion culture). In addition, static culture, aerobic culture, agitation culture, shaking culture, or rotation culture may be used, but static culture is preferred.

細胞塊の融合は、例えば、培養器材の細胞接着可能な面上で行われるとよいが、異種構造体の細胞接着可能な面上で行われてもよい。異種構造体の「異種」とは、細胞塊と由来を異にするという意味であり、細胞塊と由来を異にする構造体は、生体機能又はそれに類似する機能を有するものであるとよく、細胞塊と由来の異なる生物に由来する生体組織や臓器、人工組織や臓器などを例示することができ、管腔構造を有するものが好ましい。異種構造体としては、血管、胆管、腸管、食道、膵管、気管、尿管、卵管などの生体組織や臓器、人工血管、人工気管、細胞シートなどの人工組織や臓器、シート状構造体、チューブ状構造体、糸状構造体などを例示することができるが、これらに限定されるわけではない。異種構造体は、生体適合性の材料で形成されているとよい。生体適合性の材料としては、金属（例えば、ステンレス鋼、コバルト合金、チタン合金など）、ガラス、セラミック、合成高分子（例えば、ナイロン、ポリプロピレン、ポリジオキサノン、ポリ乳酸、ポリエチレンテレフタレート、テフロン（登録商標）など）、生体由来材料（例えば、絹、コラーゲン、脱細胞組織）などを例示することができるが、これらに限定されるわけではない。細胞接着可能な面は上述の通りである。
細胞塊の融合のための培養温度は、特に限定されないが、25～37℃とするのが好ましい。
細胞塊の融合のための培養期間は、特に限定されないが、1～10日とするのが好ましい。Fusion of cell clusters may be performed, for example, on the cell-adhesive surface of the cultureware, but may also be performed on the cell-adhesive surface of the heterogeneous structure. The "heterologous" of the heterologous structure means that the origin is different from that of the cell mass, and the structure that is different in origin from the cell mass preferably has a biological function or a function similar thereto, Examples include biological tissues and organs derived from organisms different in origin from cell aggregates, artificial tissues and organs, etc., and those having a tubular structure are preferred. Heterogeneous structures include biological tissues and organs such as blood vessels, bile ducts, intestinal tracts, esophagus, pancreatic ducts, trachea, ureters, and fallopian tubes; artificial tissues and organs such as artificial blood vessels, artificial trachea, and cell sheets; Tubular structures, filamentous structures, and the like can be exemplified, but are not limited to these. The heterostructure may be made of a biocompatible material. Biocompatible materials include metals (e.g., stainless steel, cobalt alloys, titanium alloys, etc.), glass, ceramics, synthetic polymers (e.g., nylon, polypropylene, polydioxanone, polylactic acid, polyethylene terephthalate, Teflon (registered trademark) etc.), bio-derived materials (eg, silk, collagen, decellularized tissue), etc., but are not limited to these. The cell-adherable surface is as described above.
The culture temperature for fusion of cell clusters is not particularly limited, but is preferably 25 to 37°C.
The culture period for fusion of cell clusters is not particularly limited, but is preferably 1 to 10 days.

本発明により、マトリゲルのような支持体を用いることなく、大型の細胞塊を形成することができる。
本発明の方法により、φ１００μｍ以上、φ１ｍｍ以上、φ２ｍｍ以上、φ２．５ｍｍ以上、φ４ｍｍ以上、φ６ｍｍ以上、φ８ｍｍ以上の融合細胞塊を作製することができる。φ１００μｍ以上、φ１ｍｍ以上、φ２ｍｍ以上、φ２．５ｍｍ以上、φ４ｍｍ以上、φ６ｍｍ以上、φ８ｍｍ以上の融合細胞塊は、それぞれ、大きさ８０～１５０μｍ程度の細胞塊2～4個、150～200個、300～400個、350～500個、600～800個、1200～1600個、2400～2800個から作製しうる。
また、本発明の方法で作製した融合細胞塊は、血管網構造を形成しうる。後述の実施例では、融合型肝芽に血管網構造が形成された。血管網構造としては、毛細血管、細小動静脈、類洞などが挙げられる。また、融合型肝芽は胆管構造を形成しうる。
本発明の方法により融合させた細胞塊は、融合前の細胞塊よりも機能が向上しうる。例えば、細胞塊が肝芽である場合、融合型肝芽は、融合させる前の肝芽よりも、肝分化マーカー（例えば、FoxA2、AFP、CYP3A7、CYP7A1）の遺伝子発現レベルが上昇しうる。また、従来法肝芽よりも肝機能が向上しうる。例えば、融合型肝芽は従来法肝芽よりも、肝分化マーカー（例えばALB, OTC, CYP3A7, GLUT2）の遺伝子発現レベル、アルブミン産生量・トランスフェリン産生量、アンモニア代謝量が上昇しうる。また、融合させた細胞塊を生体に移植した場合、融合させる前の細胞塊よりも、生着率が向上しうる。さらに、融合細胞塊が血管網を有する場合、生体に移植した融合細胞塊の血管網はホスト血管との吻合及び血管灌流が観察されうる。According to the present invention, large cell clusters can be formed without using a support such as Matrigel.
By the method of the present invention, a fused cell mass with a diameter of 100 μm or more, φ1 mm or more, φ2 mm or more, φ2.5 mm or more, φ4 mm or more, φ6 mm or more, or φ8 mm or more can be produced. Fused cell clusters of φ100 μm or more, φ1 mm or more, φ2 mm or more, φ2.5 mm or more, φ4 mm or more, φ6 mm or more, and φ8 mm or more are 2 to 4, 150 to 200, and 300 cell clusters with a size of about 80 to 150 μm, respectively. ~400, 350-500, 600-800, 1200-1600, 2400-2800 can be made.
In addition, the fused cell mass produced by the method of the present invention can form a vascular network structure. In Examples described later, a vascular network structure was formed in the fused liver buds. Vascular network structures include capillaries, arterioles and veins, and sinusoids. Also, fused liver buds can form bile duct structures.
A cell cluster fused by the method of the present invention may have improved functions compared to the cell cluster before fusion. For example, when the cell mass is a hepatic bud, the fused hepatic bud may have higher gene expression levels of hepatic differentiation markers (eg, FoxA2, AFP, CYP3A7, CYP7A1) than the unfused hepatic bud. In addition, liver function can be improved more than conventional liver buds. For example, fusion-type liver buds can increase gene expression levels of hepatic differentiation markers (eg, ALB, OTC, CYP3A7, GLUT2), albumin production, transferrin production, and ammonia metabolism compared to conventional liver buds. In addition, when the fused cell mass is transplanted into a living body, the engraftment rate can be improved compared to the cell mass before being fused. Furthermore, when the fused cell mass has a vascular network, the vascular network of the fused cell mass transplanted into the living body can be observed to anastomosis with the host blood vessel and vascular perfusion.

融合細胞塊をヒト又は非ヒト動物に移植することにより、例えば、移植された組織や臓器に血管網が構築され、血液灌流が開始し、高度に秩序だった組織構造を有する組織や臓器が創出されうる。よって、融合細胞塊を非ヒト動物に移植して、組織又は臓器を作製することができる。また、融合細胞塊をヒト又は非ヒト動物に移植して、組織又は臓器の再生又は機能回復を行うことができる。移植の対象となる動物は、ヒトの他、実験動物、愛玩動物、使役動物、競走馬、闘犬などに利用される動物、具体的には、マウス、ラット、ウサギ、ブタ、イヌ、サル、ウシ、ウマ、ヒツジ、ニワトリ、サメ、エイ、ギンザメ、サケ、エビ、カニなどの非ヒト動物を挙げることができる。また、非ヒト動物は、免疫拒絶反応を回避するために、免疫不全動物であるとよい。融合細胞塊の移植部位は、移植可能であればどの部位であってもよいが、血管、頭蓋内、腸間膜、肝臓、脾臓、腎臓、腎被膜下、門脈上などを例示することができる。融合細胞塊を生体（ヒト、非ヒト動物など）に移植することにより、機能が喪失あるいは低下した組織や臓器を再生させることができ、また、非ヒト動物中でヒト組織や臓器を作製させれば、創薬スクリーニングに利用することができる。 By transplanting the fused cell mass into a human or non-human animal, for example, a vascular network is constructed in the transplanted tissue or organ, blood perfusion is initiated, and a tissue or organ having a highly ordered tissue structure is created. can be Therefore, the fused cell mass can be transplanted into a non-human animal to produce a tissue or organ. Also, the fused cell mass can be transplanted into a human or non-human animal to regenerate or restore function of a tissue or organ. Animals to be transplanted include not only humans, but also animals used for laboratory animals, pets, working animals, race horses, fighting dogs, etc. Specifically, mice, rats, rabbits, pigs, dogs, monkeys, and cows. , horses, sheep, chickens, sharks, rays, chimaeras, salmon, shrimp, crabs, and other non-human animals. Also, the non-human animal is preferably an immunodeficient animal in order to avoid immune rejection. The site for transplantation of the fused cell mass may be any site as long as it can be transplanted. can. By transplanting fused cell masses into living organisms (humans, non-human animals, etc.), it is possible to regenerate tissues and organs that have lost or deteriorated in function, and to generate human tissues and organs in non-human animals. For example, it can be used for drug discovery screening.

あるいはまた、融合細胞塊をin vitroで培養して、機能をさらに向上をさせ、これを臓器類似体あるいは臓器として、ヒューマン・バイオロジー、再生医療、創薬スクリーニングなどに利用してもよい。
本発明においては、細胞塊を融合させることにより大型の血管化オルガノイド（器官芽）を創出することができ、大型化により、細胞塊の機能が向上し、生体内血管に類似した血管系（毛細血管、細小動静脈など）が形成される。細胞塊が肝芽の場合、胆管構造も形成しうる。後述の実施例では、肝細胞、間葉系細胞及び血管細胞から形成された肝芽をin vitroで融合することで、血管網構造及び／又は胆管構造が形成された。よって、本発明は、肝細胞、間葉系細胞及び血管細胞から形成された肝芽をin vitroで融合することで作製され、血管網構造及び／又は胆管構造を有する人工臓器を提供する。本発明の人工臓器において、肝細胞、間葉系細胞及び血管細胞の少なくとも１つが、人工的に作製された多能性幹細胞から分化誘導されたものであってもよい。Alternatively, the fused cell mass may be cultured in vitro to further improve its function, and used as an organ analogue or organ for human biology, regenerative medicine, drug discovery screening, and the like.
In the present invention, by fusing cell clusters, large vascularized organoids (organ buds) can be created. blood vessels, arterioles and veins, etc.) are formed. Bile duct structures may also form when the cell mass is a liver bud. In the examples described below, a vascular network structure and/or bile duct structure was formed by fusing in vitro liver buds formed from hepatocytes, mesenchymal cells and vascular cells. Accordingly, the present invention provides an artificial organ having a vascular network structure and/or bile duct structure, which is produced by in vitro fusing liver buds formed from hepatocytes, mesenchymal cells and vascular cells. In the artificial organ of the present invention, at least one of hepatocytes, mesenchymal cells and vascular cells may be differentiation-induced from artificially produced pluripotent stem cells.

融合細胞塊を非ヒト動物に移植し、生着させることにより、非ヒトキメラ動物を作製することができる。融合細胞塊を移植した非ヒト動物（例えば、マウス）は、細胞塊の由来生物種（例えば、ヒト）の生理機能を模倣しうる。 A non-human chimeric animal can be produced by transplanting the fused cell mass into a non-human animal and allowing it to engraft. Non-human animals (eg, mice) implanted with fused cell masses can mimic the physiology of the species from which the cell masses are derived (eg, humans).

さらに、融合細胞塊、融合細胞塊から作製された組織及び臓器、及び融合細胞塊を移植した非ヒトキメラ動物からなる群より選択される少なくとも１つを用いて、薬剤を評価することができる。薬剤の評価としては、例えば、薬物代謝の評価（例えば、薬物代謝プロファイルの予測）、薬効評価（例えば、医薬品として有効な薬剤をスクリーニングすることなど）、毒性評価、薬物相互作用評価などを挙げることができる。 Furthermore, the drug can be evaluated using at least one selected from the group consisting of fused cell masses, tissues and organs prepared from fused cell masses, and non-human chimeric animals transplanted with fused cell masses. Examples of drug evaluation include evaluation of drug metabolism (e.g., prediction of drug metabolism profile), drug efficacy evaluation (e.g., screening of effective drugs as drugs), toxicity evaluation, drug interaction evaluation, and the like. can be done.

薬物代謝の評価は、ヒト又は非ヒト動物由来の細胞から作製された細胞塊を融合して得られた融合細胞塊、融合細胞塊から作製された組織及び臓器、又は融合細胞塊を移植した非ヒトキメラ動物から、医薬品の候補化合物を投与したのちの生物試料を採取・解析することにより、ヒト型の薬物代謝プロファイルを取得することができる。これにより従来の技術では達成が極めて難しかったヒトでの医薬品の分布・代謝・***過程を予測することが可能となり、安全で効果のある医薬品の開発を飛躍的に加速できるものと期待される。 Evaluation of drug metabolism should be performed in fused cell masses obtained by fusing cell masses prepared from cells derived from human or non-human animals, tissues and organs prepared from fused cell masses, or non-transplanted fused cell masses. A human-type drug metabolism profile can be obtained by collecting and analyzing a biological sample after administration of a drug candidate compound from a human chimeric animal. This makes it possible to predict the distribution, metabolism, and excretion processes of drugs in humans, which was extremely difficult to achieve with conventional technology, and is expected to dramatically accelerate the development of safe and effective drugs.

医薬品として有効な薬剤のスクリーニングは、ヒト又は非ヒト動物由来の細胞から作製された細胞塊を融合して得られた融合細胞塊、融合細胞塊から作製された組織及び臓器、又は融合細胞塊を移植した非ヒトキメラ動物に、新規の医薬品候補化合物を投与することにより解析することが可能となる。これにより、従来のｉｎ ｖｉｔｒｏ試験で不十分であった、実際ヒトに投与した際の薬効の予測精度を大幅に改善できる可能性が期待される。 Screening of drugs that are effective as pharmaceuticals is conducted by fusing cell clusters prepared from human or non-human animal-derived cells, tissues and organs prepared from the fused cell clusters, or fused cell clusters. Analysis can be performed by administering a novel drug candidate compound to the transplanted non-human chimeric animal. As a result, it is expected that the accuracy of predicting drug efficacy when actually administered to humans, which was insufficient in conventional in vitro tests, can be greatly improved.

毒性評価は、ヒト又は非ヒト動物由来の細胞から作製された細胞塊を融合して得られた融合細胞塊、融合細胞塊から作製された組織及び臓器、又は融合細胞塊を移植した非ヒトキメラ動物に被験物質を投与したのちに、組織障害マーカーなどを測定することにより、障害予測の精度向上が可能となる。 Toxicity evaluation is performed on fused cell clusters obtained by fusing cell clusters prepared from cells derived from human or non-human animals, tissues and organs prepared from fused cell clusters, or non-human chimeric animals transplanted with fused cell clusters. By measuring tissue damage markers and the like after administration of the test substance, it is possible to improve the accuracy of damage prediction.

薬物相互作用評価は、ヒト又は非ヒト動物由来の細胞から作製された細胞塊を融合して得られた融合細胞塊、融合細胞塊から作製された組織及び臓器、又は融合細胞塊を移植した非ヒトキメラ動物に、複数の薬剤を投与したのちに、その後の各薬剤の分布・代謝・***過程などの薬物動態や毒性評価、薬効評価を行うことにより行うことができる。 The evaluation of drug interactions is performed on fused cell masses obtained by fusing cell masses made from cells derived from human or non-human animals, tissues and organs made from fused cell masses, or non-human cells transplanted with fused cell masses. It can be carried out by administering a plurality of drugs to a human chimeric animal, and then conducting pharmacokinetics such as distribution, metabolism, and excretion processes of each drug, toxicity evaluation, and drug efficacy evaluation.

本発明の方法により作製した融合細胞塊は、再生医療用組成物の有効成分として利用することができる。 A fused cell mass produced by the method of the present invention can be used as an active ingredient of a composition for regenerative medicine.

再生医療用組成物をヒト又は非ヒト動物の生体内に移植し、組織又は臓器を作製することができる。また、再生医療用組成物を生体内に移植し、組織又は臓器の再生又は機能回復を行うことができる。 A composition for regenerative medicine can be transplanted into a human or non-human animal body to produce a tissue or an organ. In addition, the composition for regenerative medicine can be transplanted into a living body to regenerate or restore the function of a tissue or organ.

再生医療用組成物がヒト又は非ヒト動物の生体に移植された後、融合細胞塊は血管網を有する組織又は臓器に分化しうる。その血管網には血液灌流が生じうる。血管網に血液灌流が生じることにより、成体組織と同等若しくはそれに近い高度に秩序だった組織構造を有する組織・臓器を創出することが可能となると考えられる。 After the composition for regenerative medicine is transplanted into a human or non-human animal body, the fused cell mass can differentiate into a tissue or organ having a vascular network. Blood perfusion can occur in the vascular network. It is thought that it will be possible to create tissues and organs that have a highly ordered tissue structure equivalent to or close to that of adult tissues by causing blood perfusion in the vascular network.

再生医療用組成物には、ＦＧＦ２、ＨＧＦ、ＶＥＧＦなどの組織血管化促進剤、移植に伴う止血用ゼラチンスポンジ（商品名：スポンゼル、アステラス株式会社）、および、移植組織の固定に用いるボルヒール（帝人ファーマ株式会社）・ベリプラスト（ＣＳＬベーリング株式会社）・タココンブ（ＣＳＬベーリング株式会社）、コラーゲン、マトリゲルなどの組織接着剤などを添加してもよい。
Compositions for regenerative medicine include tissue vascularization promoters such as FGF2, HGF, and VEGF, gelatin sponges for hemostasis associated with transplantation (trade name: Sponzel, Astellas Co., Ltd.), and Borheal (Teijin Limited) for fixing transplanted tissues. Pharma Co., Ltd.), Veriplast (CSL Behring Co., Ltd.), Octopus kombu (CSL Behring Co., Ltd.), collagen, tissue adhesives such as matrigel, and the like may be added.

以下、実施例により本発明を更に詳細に説明する。
〔実施例１〕
実験方法
・ヒト人工多能性幹細胞(iPSC)の培養法
細胞培養ディッシュあるいは細胞培養プレートをiMatrix-511 (Nippi, 0.7～0.9 μg/cm2)で37℃、１時間コーティングし、PBSで洗浄した。凍結保存ヒトiPSC（TkDA株又は1231A3株、それぞれ東京大学、京都大学より入手）を37℃のお湯に２分間浸漬し、手で振盪しながら融解させた。細胞保存液の９倍量のStemFit培地（Ajinomoto）に細胞保存液を懸濁し、150-200 x g, 5分間の遠心操作を行った。細胞上清を除き、AK02培地にY-27632 (10 uM)を加えた培地に細胞を懸濁し、0.36～1.8 x 103 cells/cm2の濃度でヒトiPSCを播種した。培養１日目にAK02培地に交換し、以降１日おきに培地交換を行った。継代に関しては、直径１０cmの細胞培養ディッシュで一週間培養したヒトiPSCをPBSで洗浄した後、Accutase 2 mlを加え37℃で5分から１０分間処理し、細胞を剥離した。AK02培地2 mlを加えて、15 mlのチューブに細胞を移し、150-200 x g, 5 minの遠心操作を行った。細胞上清を除き、AK02培地にY-27632 (10 uM)を加えた培地に細胞を懸濁し、0.36～1.8 x 103 cells/cm2の濃度でヒトiPSCを播種した。EXAMPLES The present invention will be described in more detail below with reference to examples.
[Example 1]
Experimental method ・Human induced pluripotent stem cell (iPSC) culture method A cell culture dish or cell culture plate was coated with iMatrix-511 (Nippi, 0.7-0.9 μg/cm 2 ) at 37°C for 1 hour and washed with PBS. . Cryopreserved human iPSCs (TkDA strain or 1231A3 strain, obtained from the University of Tokyo and Kyoto University, respectively) were immersed in hot water at 37°C for 2 minutes and thawed while shaking by hand. The cell preservation medium was suspended in StemFit medium (Ajinomoto) 9 times the amount of the cell preservation medium, and centrifuged at 150-200 xg for 5 minutes. The cell supernatant was removed, the cells were suspended in a medium containing AK02 medium and Y-27632 (10 uM), and human iPSCs were seeded at a concentration of 0.36 to 1.8 x 103 cells/ cm2 . On the first day of culture, the medium was changed to AK02 medium, and thereafter the medium was changed every other day. For passage, human iPSCs cultured in a cell culture dish of 10 cm in diameter for one week were washed with PBS, then added with 2 ml of Accutase and treated at 37° C. for 5 to 10 minutes to detach the cells. 2 ml of AK02 medium was added, the cells were transferred to a 15 ml tube, and centrifuged at 150-200 xg for 5 min. The cell supernatant was removed, the cells were suspended in a medium containing AK02 medium and Y-27632 (10 uM), and human iPSCs were seeded at a concentration of 0.36 to 1.8 x 103 cells/ cm2 .

・iPSCからの肝前駆細胞への分化誘導法
細胞培養ディッシュあるいは細胞培養プレート（Falcon）をiMatrix-511 (Nippi, 0.4～0.6 μg/cm2)で37℃、１時間コーティングし、PBSで洗浄した。直径１０cmの細胞培養ディッシュで一週間培養したヒトiPSCをPBS洗浄後にAccutase 2 mlを加え37℃で5分から１０分間処理し、細胞を剥離した。細胞回収・遠心後に細胞上清を除き、RPMI培地にペニシリン/ストレプトマイシン（1%）、B27 (2%)、Wnt3a (50 ng/ml)、Activin A (100 ng/ml)、Y-27632 (10 uM)を加えた培地に細胞を懸濁し、ラミニンコートしたディッシュに5～10 x 104 cells/cm2の密度で播種した。培養１日目と３日目に、RPMI培地にペニシリン/ストレプトマイシン（1%）、B27 (2%)、Wnt3a (50 ng/ml)、Activin A (100 ng/ml)、Sodium Butyrate (0.5 mM)を加えた培地に交換した。培養４日目にRPMI培地にペニシリン/ストレプトマイシン（1%）、B27 (2%)、Wnt3a (50 ng/ml)、Activin A (100 ng/ml) を加えた培地に交換した。培養６日目の細胞を、内胚葉細胞Definitive Endoderm (DE)とした。培養６日目と培養８日目に、RPMI培地にペニシリン/ストレプトマイシン（1%）B27 (2%)、basic FGF (10 ng/ml)、BMP-4 (20 ng/ml)を加えた培地に交換した。培養１０日目の細胞を、肝前駆体細胞Hepatic Endoderm (HE)とした。・Method of inducing differentiation from iPSCs to hepatic progenitor cells A cell culture dish or cell culture plate (Falcon) was coated with iMatrix-511 (Nippi, 0.4-0.6 μg/cm 2 ) at 37°C for 1 hour and washed with PBS. . Human iPSCs cultured in a cell culture dish with a diameter of 10 cm for one week were washed with PBS, added with 2 ml of Accutase and treated at 37° C. for 5 to 10 minutes to detach the cells. After cell collection and centrifugation, cell supernatant was removed, and penicillin/streptomycin (1%), B27 (2%), Wnt3a (50 ng/ml), Activin A (100 ng/ml), Y-27632 (10 uM), and seeded on a laminin-coated dish at a density of 5 to 10 x 104 cells/ cm2 . Penicillin/streptomycin (1%), B27 (2%), Wnt3a (50 ng/ml), Activin A (100 ng/ml), Sodium Butyrate (0.5 mM) were added to RPMI medium on days 1 and 3 of culture. was replaced with a medium containing On day 4 of culture, the RPMI medium was replaced with a medium supplemented with penicillin/streptomycin (1%), B27 (2%), Wnt3a (50 ng/ml) and Activin A (100 ng/ml). Cells on day 6 of culture were used as endoderm cells Definitive Endoderm (DE). On day 6 and day 8 of culture, penicillin/streptomycin (1%) B27 (2%), basic FGF (10 ng/ml), and BMP-4 (20 ng/ml) were added to RPMI medium. exchanged. Cells on day 10 of culture were used as hepatic progenitor cells Hepatic Endoderm (HE).

・iPSCからの血管内皮細胞への分化誘導法
iPSCからの血管内皮細胞への分化誘導は既報に従って行った（Takebe et al., Cell Reports, 2017）。概要を説明すると、肝前駆細胞と同様にiPSCを播種し、StemFit培地にY-27632 (10 uM)を加えた培地で1日培養を行った。翌日にDMEM/F12培地に1% Glutamax、1% B27、CHIR99021 (8 uM)、BMP-4 (25 ng/ml)を加えた培地に交換し、3日間培養後にStemPro-34 SFM培地にVEGF (200 ng/ml)、forskolin (2 uM)を加えた培地に交換した。分化誘導開始から7日目にFACSによりCD31やCD144の発現を確認することで品質のチェックを行った。得られた血管内皮細胞は、StemPro-34 SFM培地にVEGF (50 ng/ml)を加えた培地を用いて、フィブロネクチンコートした培養ディッシュ上で継代し、拡大培養を行った。・Method of inducing differentiation from iPSCs to vascular endothelial cells
Differentiation of iPSCs into vascular endothelial cells was induced according to a previous report (Takebe et al., Cell Reports, 2017). Briefly, iPSCs were seeded in the same manner as hepatic progenitor cells and cultured for 1 day in StemFit medium plus Y-27632 (10 uM). The next day, the medium was changed to DMEM/F12 medium supplemented with 1% Glutamax, 1% B27, CHIR99021 (8 uM), and BMP-4 (25 ng/ml). After 3 days of culture, VEGF ( 200 ng/ml) and forskolin (2 uM). The quality was checked by confirming the expression of CD31 and CD144 by FACS on the 7th day from the start of differentiation induction. The obtained vascular endothelial cells were subcultured and expanded on a fibronectin-coated culture dish using a StemPro-34 SFM medium supplemented with VEGF (50 ng/ml).

・iPSCからの間葉系細胞への分化誘導法
iPSCからの間葉系細胞への分化誘導は既報に従って行った（Takebe et al., Cell Reports, 2017）。概要を説明すると、肝前駆細胞と同様2-8 x 10^3 cells/cm2の密度でiPSCを播種し、StemFit培地にY-27632 (10 uM)を加えた培地で4-6日培養を行った。DMEM/F12培地に1% Glutamax、1% B27、CHIR99021 (8 uM)、BMP-4 (25 ng/ml)を加えた培地に交換し、3日間培養後にDMEM/F12培地に1% Glutamax、1% B27、Activin A (2 ng/ml)、PDGFBB (10 ng/ml)を加えた培地に交換した。３日後にStemPro-34 SFM培地にFGF2 (10 ng/ml)、PDGFBB (10 ng/ml)を加えた培地に交換し３日間培養を行った。・Method for inducing differentiation from iPSCs to mesenchymal cells
Induction of differentiation from iPSCs to mesenchymal cells was performed according to a previous report (Takebe et al., Cell Reports, 2017). Briefly, iPSCs were seeded at a density of 2-8 x 10^3 cells/ cm2 , similar to hepatic progenitor cells, and cultured for 4-6 days in StemFit medium plus Y-27632 (10 uM). gone. Change the medium to DMEM/F12 medium supplemented with 1% Glutamax, 1% B27, CHIR99021 (8 uM), and BMP-4 (25 ng/ml). % B27, Activin A (2 ng/ml), and PDGFBB (10 ng/ml) were added to replace the medium. After 3 days, the StemPro-34 SFM medium was replaced with a medium containing FGF2 (10 ng/ml) and PDGFBB (10 ng/ml), and cultured for 3 days.

・ヒト間葉系幹細胞(MSC)の培養法
MSCGM培地（Lonza）に懸濁したMesenchymal Stem cells (以下MSCと記載, Lonza, 3-5 x 10^5 cells)を、細胞培養ディッシュ (直径10ｃｍ)に播種した。3日に１回培地交換を行った。７日後にPBSで洗浄後、2 mlのTrypsin/EDTA (Gibco)を加え37℃で5分処理し、細胞剥離を行った。MSCGM培地を2 ml加えて、15 mlのチューブに細胞を移し、150-200 x g, 5 minの遠心操作を行った。細胞上清を除き、HCM (EGF不含)培地 (Lonza)にFBS Gold (MP Biomedicals) 5%、HGF 10 ng/ml、Oncostatin M (R&D) 20 ng/ml、Dexamethazon 100 nMを添加した培地とEGM培地を１：１の割合で混合した培地(HCM/EGM混合培地)に細胞を懸濁し、肝芽の作製に供した。・Method for culturing human mesenchymal stem cells (MSCs)
Mesenchymal stem cells (hereinafter referred to as MSC, Lonza, 3-5 x 10^5 cells) suspended in MSCGM medium (Lonza) were seeded in a cell culture dish (10 cm in diameter). The medium was replaced once every 3 days. After 7 days, the cells were washed with PBS, added with 2 ml of Trypsin/EDTA (Gibco), and treated at 37°C for 5 minutes to detach the cells. 2 ml of MSCGM medium was added, the cells were transferred to a 15 ml tube, and centrifuged at 150-200 xg for 5 min. Cell supernatant was removed, and HCM (EGF-free) medium (Lonza) supplemented with FBS Gold (MP Biomedicals) 5%, HGF 10 ng/ml, Oncostatin M (R&D) 20 ng/ml, Dexamethazon 100 nM and Cells were suspended in a medium mixed with EGM medium at a ratio of 1:1 (HCM/EGM mixed medium) and subjected to production of liver buds.

・ヒト臍帯静脈由来血管内皮細胞（HUVEC）の培養法
EGM培地(Lonza)又はEGM-2培地(Lonza)に懸濁したKusabira Orange標識Human Umbilical Vein Endothelial Cells (HUVECと記載, Lonza, 3-5 x 10^5 cells)を、細胞培養ディッシュ (直径10ｃｍ)に播種した。3日に１回培地交換を行った。７日後にPBSで洗浄後、2 mlのTrypsin/EDTA (Gibco)を加え37℃で5分処理し、細胞剥離を行った。EGM培地又はEGM-2培地を2 ml加えて、15 mlのチューブに細胞を移し、150-200 x g, 5分の遠心操作を行った。細胞上清を除き、HCM/EGM混合培地に細胞を懸濁し、肝芽の作製に供した。・Culturing method of human umbilical vein-derived vascular endothelial cells (HUVEC)
Kusabira Orange-labeled Human Umbilical Vein Endothelial Cells (HUVEC, Lonza, 3-5 x 10^5 cells) suspended in EGM medium (Lonza) or EGM-2 medium (Lonza) were placed in a cell culture dish (10 cm in diameter). sown in The medium was replaced once every 3 days. After 7 days, the cells were washed with PBS, added with 2 ml of Trypsin/EDTA (Gibco), and treated at 37°C for 5 minutes to detach the cells. 2 ml of EGM medium or EGM-2 medium was added, the cells were transferred to a 15 ml tube, and centrifuged at 150-200 xg for 5 minutes. After removing the cell supernatant, the cells were suspended in HCM/EGM mixed medium and subjected to production of liver buds.

・肝芽作製法
小型肝芽の作製に関しては、マイクロパターンウェルプレートElplasia RB 500 400 NA (クラレ)24ウェルプレート１ウェル当たりヒトiPSC由来DE又はHE 2.3-5 x 105 cellsとHUVEC又はiPSC-EC 0.5-3.5 x 105 cellsとMSC又はiPSC-MC 0.5-3.5 x 105 cellsをHCM/EGM混合培地に懸濁し播種した。従来法に関しては、48ウェルプレート１ウェル当たりヒトiPSC由来DE又はHE 5 x 105 cellsとHUVEC又はiPSC-EC 3.5 x 105 cellsとMSC又はiPSC-MC 0.5 x 105 cellsをHCM/EGM混合培地に懸濁し、予め37℃で固化した50%マトリゲル (Matrigel: EGM=1:1, 150 ul/well, 48 well plate)上に播種した。肝芽は37℃、5% CO2環境下で24時間培養を行い、肝芽融合に用いた。・Method for producing small liver buds For the production of small liver buds, human iPSC-derived DE or HE 2.3-5 x 10 5 cells and HUVEC or iPSC-EC per well of micropatterned well plate Elplasia RB 500 400 NA (Kuraray) 24-well plate 0.5-3.5×10 5 cells and 0.5-3.5×10 5 cells of MSCs or iPSC-MCs were suspended in HCM/EGM mixed medium and seeded. For the conventional method, human iPSC-derived DE or HE 5 x 10 5 cells and HUVEC or iPSC-EC 3.5 x 10 5 cells and MSC or iPSC-MC 0.5 x 10 5 cells per well of a 48-well plate were mixed in HCM/EGM mixed medium. and seeded on 50% Matrigel (Matrigel: EGM=1:1, 150 ul/well, 48 well plate) previously solidified at 37°C. Liver buds were cultured at 37°C and 5% CO 2 for 24 hours and used for liver bud fusion.

・肝芽融合法
肝芽作製24時間後、マイクロパターンウェルプレートElplasia RB 500 400 NA (クラレ)より小型肝芽を回収した。回収した小型肝芽を24ウェルセルカルチャーインサート（Falcon）、12ウェルセルカルチャーインサート（Falcon）、6ウェルセルカルチャーインサート（Falcon）上、あるいはPrimeSurface（登録商標）プレート96U、PrimeSurface（登録商標）プレート96V（住友ベークライト）、超低接着表面プレート９６ウェル平底フタ付きまたは96ウェル透明細胞培養表面処理ポリスチレンマイクロプレート（コーニング）、PTFE製人工血管C-Porous多孔質チューブ（中興化成工業）に肝芽同士を近接させ配置した。24ウェルセルカルチャーインサート（Falcon）を使用する際には、HCM/EGM混合培地500 ul/wellとし、小型肝芽600個または従来型肝芽２個を配置した。12ウェルセルカルチャーインサート（Falcon）を使用する際には、HCM/EGM混合培地1 ml/wellとし、小型肝芽1200個を配置した。6ウェルセルカルチャーインサート（Falcon）を使用する際には、HCM/EGM混合培地2 ml/wellとし、小型肝芽2400個を配置した。セルカルチャーインサート上にシリコン枠（Culture-Insert 2well/3well, ibidi）を使用することも可能である。シリコン枠を使用したのは図３，９，１０である。インサート表面はコラーゲンIでコーティングを行った。人工血管表面は0.1% ゼラチンコートを行った。PrimeSurface（登録商標）プレート96U、PrimeSurface（登録商標）プレート96V（住友ベークライト）、超低接着表面プレート９６ウェル平底フタ付きまたは96ウェル透明細胞培養表面処理ポリスチレンマイクロプレート（コーニング）を使用する際には、HCM/EGM混合培地200 ul/wellとし、小型肝芽600個または従来型肝芽２個を配置した。小型肝芽の密度はいずれも30000 spheroids/mlである。その後、37℃、5% CO2環境下で７日間培養を行った。配置直後、配置後１日、３日、７、９日後に蛍光顕微鏡BZ-X710（Keyence）、共焦点顕微鏡SP5又はSP8を用いて写真撮影を行った。培養９日後に４％PFA/PBSにて固定し、パラフィン包埋後に７～１０ μmの切片を作製し、組織解析を行った。• Liver bud fusion method Twenty-four hours after the preparation of liver buds, small liver buds were recovered from a micropattern well plate Elplasia RB 500 400 NA (Kuraray). Collected small liver buds were plated onto 24-well cell culture inserts (Falcon), 12-well cell culture inserts (Falcon), 6-well cell culture inserts (Falcon) or PrimeSurface® Plate 96U, PrimeSurface® Plate 96V. (Sumitomo Bakelite), 96-well flat-bottom lidded ultra-low attachment surface plate or 96-well clear cell culture surface-treated polystyrene microplate (Corning), PTFE artificial blood vessel C-Porous porous tube (Chuko Kasei Kogyo) between liver buds. placed close together. When using a 24-well cell culture insert (Falcon), HCM/EGM mixed medium was 500 ul/well, and 600 small liver buds or 2 conventional liver buds were arranged. When using a 12-well cell culture insert (Falcon), 1 ml/well of HCM/EGM mixed medium was used and 1200 small liver buds were arranged. When using a 6-well cell culture insert (Falcon), 2400 small liver buds were placed in 2 ml/well of HCM/EGM mixed medium. It is also possible to use a silicon frame (Culture-Insert 2well/3well, ibidi) on the cell culture insert. 3, 9 and 10 use a silicon frame. The insert surface was coated with collagen I. The artificial blood vessel surface was coated with 0.1% gelatin. When using PrimeSurface® Plate 96U, PrimeSurface® Plate 96V (Sumitomo Bakelite), Ultra Low Attachment Surface Plate 96-well Flat Bottom Lid or 96-well Clear Cell Culture Surface Treated Polystyrene Microplate (Corning) , HCM/EGM mixed medium 200 ul/well, and 600 small liver buds or 2 conventional liver buds were arranged. The density of small liver buds is 30000 spheroids/ml. After that, the cells were cultured for 7 days at 37°C in a 5% CO 2 environment. Photographs were taken using a fluorescence microscope BZ-X710 (Keyence) and a confocal microscope SP5 or SP8 immediately after placement and 1 day, 3 days, 7 and 9 days after placement. After 9 days of culture, the cells were fixed with 4% PFA/PBS, embedded in paraffin, and sectioned at 7 to 10 μm for tissue analysis.

・クラニアルウィンドウへの肝芽移植
免疫不全マウスNOD/Scidは、三共ラボサービス株式会社より購入した。NOD/Scidマウスの腹腔内にケタミン/キシラジン混合麻酔薬あるいは塩酸メデトミジン/ミゾダラム/ブトルファノール混合液を投与した後、頭蓋骨を歯科用ドリルで円形に掘削し取り除いた。硬膜を取り除いた後生理食塩水を加え、特注円形ガラス(Matsunami)を乗せて歯科材料 (COE TRAY PLASTIC)で封入を行った。１週間後ガラスを除き、小型肝芽又は融合型肝芽を脳表面上に移植し封入した。移植後は、経時的に蛍光実体顕微鏡（Carl Zeiss）又は共焦点顕微鏡SP5（Leica）又はSP8（Leica）を用いて写真撮影を行った。血流を観察する際にはAngiosense 680 (PerkinElmer)を尾静脈に注射した。移植３週後に移植片を４％PFA/PBSにて固定し、パラフィン包埋後に７～１０ μmの切片を作製し、組織解析を行った。- Transplantation of liver buds to cranial window Immunodeficient mouse NOD/Scid was purchased from Sankyo Labo Service Co., Ltd. NOD/Scid mice were intraperitoneally administered ketamine/xylazine mixed anesthetic or medetomidine hydrochloride/mizodaram/butorphanol mixed solution, and then the skull was drilled circularly with a dental drill and removed. After removing the dura mater, physiological saline was added, and a custom-made circular glass (Matsunami) was mounted and sealed with a dental material (COE TRAY PLASTIC). After one week, the glass was removed and small liver buds or fused liver buds were transplanted onto the brain surface and encapsulated. After transplantation, photographs were taken over time using a fluorescence stereomicroscope (Carl Zeiss) or a confocal microscope SP5 (Leica) or SP8 (Leica). Angiosense 680 (PerkinElmer) was injected into the tail vein when observing blood flow. Three weeks after transplantation, the graft was fixed with 4% PFA/PBS, embedded in paraffin, and sectioned at 7 to 10 μm for histological analysis.

・定量的RT-PCRを用いた融合型肝芽の機能評価
Elplasia RB 500 400 NA(クラレ)で作製した小型肝芽と融合型肝芽を培養後10日目に回収し、PureLink RNA mini kit (Thermo Fisher Scientific)を用いてRNAを精製した。cDNA合成はiScript cDNA Synthesis Kit (Bio-Rad)を用いて行い、KAPA SYBR（登録商標） Fast qPCR kit（日本ジェネティクス）とLightCycler（登録商標） 480 (Roche Life Science)を用いて遺伝子の増幅・検出を行った。・Functional evaluation of fused liver buds using quantitative RT-PCR
Small liver buds and fused liver buds prepared with Elplasia RB 500 400 NA (Kuraray) were harvested on day 10 after culturing, and RNA was purified using the PureLink RNA mini kit (Thermo Fisher Scientific). cDNA synthesis was performed using iScript cDNA Synthesis Kit (Bio-Rad), and gene amplification and amplification was performed using KAPA SYBR (registered trademark) Fast qPCR kit (Nippon Genetics) and LightCycler (registered trademark) 480 (Roche Life Science). detected.

・ELISAを用いた融合型肝芽の機能評価
融合型肝芽培養１０日目に培地交換を行ない、その24時間後に培地を回収し、培養上清中のアルブミンやトランスフェリン量を市販のELISAキット（それぞれBethyl Laboratories, Abcam）により測定した。・ Functional evaluation of fused liver buds using ELISA On the 10th day of culture of fused liver buds, the medium was changed, the medium was collected 24 hours later, and the amount of albumin and transferrin in the culture supernatant was measured using a commercially available ELISA kit ( Each was measured by Bethyl Laboratories, Abcam).

・尿素・アンモニア代謝系の機能評価
融合型肝芽培養１０日目に培地交換を行ない、その24時間後に培地を回収し、培養上清中の尿素量をQuantiChrom Urea Assay Kitにより測定した。アンモニア代謝については、培地回収後に2 mM塩化アンモニウムを含む培地に交換し、24時間後に培地回収を行った。培養上清中のアンモニア量をアミチェック（アークレイ）により測定し、2 mM塩化アンモニウムを含む培地添加直後の培地中のアンモニア量との差分をアンモニア代謝量とした。-Functional evaluation of urea/ammonia metabolism system On the 10th day of culture of the fused liver bud, the medium was exchanged, the medium was recovered 24 hours later, and the amount of urea in the culture supernatant was measured using the QuantiChrom Urea Assay Kit. Regarding ammonia metabolism, the medium was replaced with a medium containing 2 mM ammonium chloride after collecting the medium, and the medium was collected after 24 hours. The amount of ammonia in the culture supernatant was measured by Amicheck (Arkray), and the difference between the amount of ammonia in the medium immediately after the addition of the medium containing 2 mM ammonium chloride was defined as the metabolic rate of ammonia.

・パラフィン切片作製法
回収された組織は、4%パラホルムアルデヒド/リン酸緩衝液（PBS）中で１６時間固定を行った。固定組織をPBSで３回洗浄した後、70%エタノール、95%エタノール、100%エタノール（２回）にそれぞれ３０分以上浸漬し、組織の脱水を行う。その後、50%エタノール/キシレン溶液、100%キシレン溶液、100%パラフィン溶液（２回）中それぞれ65℃で３０分以上振盪しながら浸漬した後、パラフィンに包埋した。包埋した組織サンプルは、ミクロトームで7～10 μmの厚さに薄切を行った。薄切後、37度で試料の乾燥を行った。- Paraffin section preparation method The recovered tissue was fixed in 4% paraformaldehyde/phosphate buffer (PBS) for 16 hours. After the fixed tissue is washed with PBS three times, it is immersed in 70% ethanol, 95% ethanol, and 100% ethanol (twice) for 30 minutes or longer to dehydrate the tissue. After that, they were immersed in 50% ethanol/xylene solution, 100% xylene solution, and 100% paraffin solution (twice) with shaking at 65°C for 30 minutes or more, and then embedded in paraffin. Embedded tissue samples were sectioned with a microtome to a thickness of 7-10 μm. After slicing, the sample was dried at 37 degrees.

・ヘマトキシリン・エオジン染色
乾燥させたパラフィン切片をキシレン、100%エタノール、95%エタノール、70%エタノール、ミリQ水と段階を経て脱パラフィン、親水化を行った。流水で洗浄後、ヘマトキシリン溶液で３分間染色し、流水で洗浄した後、エオジン溶液で２分間染色を行う。流水で洗浄後、アルコール脱水を行い、キシレンで透徹後にサンプルに非水溶性封入剤をのせ、カバーガラスにより封入を行った。• Hematoxylin/eosin staining Dried paraffin sections were deparaffinized and hydrophilized through the steps of xylene, 100% ethanol, 95% ethanol, 70% ethanol, and milli-Q water. After washing with running water, it is stained with a hematoxylin solution for 3 minutes, and after washing with running water, it is stained with an eosin solution for 2 minutes. After washing with running water, dehydration with alcohol was performed, and after transparent with xylene, a water-insoluble mounting medium was placed on the sample, and the sample was mounted with a cover glass.

・免疫組織化学的解析
乾燥させたパラフィン切片をキシレン、100%エタノール、95%エタノール、70%エタノール、ミリQ水と段階を経て脱パラフィン、親水化を行う。10 mMクエン酸緩衝液（pH 6.0）中、121℃、15分間の抗原賦活化処理後、3% 過酸化水素水/メタノール液中１０分間処理し、内在性のペルオキシダーゼを不活性化した。Blocking One溶液（Nacalai Tesque）で室温１時間反応させた後、希釈した１次抗体（hCD31抗体（M0823, DAKO）、hAlbmin抗体（A6684, Sigma）、HNF4α抗体（sc-6556, Santa Cruz Biotechnology）、Vimentin抗体（M7020, DAKO））と室温１時間または4℃一晩反応させた。PBSで３回洗浄した後、HRP標識あるいは蛍光標識された２次抗体と室温30分以上反応させた。PBSで3回洗浄後、蛍光標識された２次抗体の場合は、DAPIによる核染色後に封入し、顕微鏡観察を行った。HRP標識された２次抗体の場合はDAB Reagent (DAKO)で２～１０分間反応させ、発色を行った。精製水で洗浄後、ヘマトキシリンで3分間染色し、10分間の流水で洗浄した後に0.1% Sodium Bicarbonateで5分間反応させ、アルコールによる脱水を行い、キシレンで透徹した後にサンプルに非水溶性封入剤をのせ、カバーガラスで覆い封入を行った。・Immunohistochemical analysis Dried paraffin sections are deparaffinized and hydrophilized in xylene, 100% ethanol, 95% ethanol, 70% ethanol, and milli-Q water. After antigen retrieval treatment in 10 mM citrate buffer (pH 6.0) at 121° C. for 15 minutes, endogenous peroxidase was inactivated by treatment in 3% hydrogen peroxide/methanol solution for 10 minutes. After reacting with Blocking One solution (Nacalai Tesque) for 1 hour at room temperature, diluted primary antibodies (hCD31 antibody (M0823, DAKO), hAlbmin antibody (A6684, Sigma), HNF4α antibody (sc-6556, Santa Cruz Biotechnology), Vimentin antibody (M7020, DAKO)) was reacted at room temperature for 1 hour or at 4°C overnight. After washing with PBS three times, it was allowed to react with HRP-labeled or fluorescence-labeled secondary antibody for 30 minutes or more at room temperature. After washing with PBS three times, in the case of a fluorescently-labeled secondary antibody, nuclear staining with DAPI was followed by encapsulation and microscopic observation. In the case of HRP-labeled secondary antibodies, they were reacted with DAB Reagent (DAKO) for 2 to 10 minutes for color development. After washing with purified water, the sample was stained with hematoxylin for 3 minutes, washed with running water for 10 minutes, reacted with 0.1% sodium bicarbonate for 5 minutes, dehydrated with alcohol, and cleared with xylene. It was placed on the plate and covered with a cover glass for encapsulation.

・播種面に対する細胞塊占有面積の算出法
細胞塊占有面積の算出には、画像解析ソフトImageJ 1.51s（National Institutes of Health, U.S.A., http://imagej.nih.gov/ijよりダウンロード可能）を用いた。細胞塊を含む全播種面を選択し、Image→Adjust→ThresholdよりColorをB&W選択し、画像を白黒に変更する。本条件により細胞塊が白、播種面が黒となる。閾値の最小値（上部）を130に、閾値の最大値（下部）を255に設定すると、細胞塊が占有する播種面以外の播種面の割合（％）がThreshold画面左のグラフ下に表示される。１００％からの差分を細胞塊占有面積（％）として算出した。・Method for calculating the area occupied by cell clusters on the seeding surface To calculate the area occupied by cell clusters, use the image analysis software ImageJ 1.51s (National Institutes of Health, USA, available for download from http://imagej.nih.gov/ij). Using. Select the entire seeding surface including the cell mass, select Color B&W from Image → Adjust → Threshold, and change the image to black and white. Under these conditions, the cell mass becomes white and the seeded surface becomes black. When the minimum threshold value (upper) is set to 130 and the maximum threshold value (lower) is set to 255, the ratio (%) of the seeding surface other than the seeding surface occupied by cell clusters is displayed under the graph on the left side of the Threshold screen. be. The difference from 100% was calculated as the cell mass occupied area (%).

実験結果
・ヒトiPSC肝芽の凝集・融合
Elplasiaプレートで作製された小型肝芽（約80～150 um）又はマトリゲル上で作製された従来法肝芽（約1～2 mm）複数個を翌日にセルカルチャーインサート、Uボトムプレート、Vボトムプレートに移動させ、肝芽の凝集及び融合を７日目まで経時的に観察した。その結果、融合１日後からセルカルチャーインサート、Uボトムプレート、Vボトムプレート、Fボトムプレート、低吸着Fボトムプレート、人工血管上での小型肝芽の凝集及び融合が観察された（図1A-F）。Fボトムプレートでは肝芽の部分的な融合が観察された。融合型肝芽の大きさは、それぞれセルカルチャーインサートで約4 mm、Uボトムプレートで約2 mm、Vボトムプレートで約2 mm、低吸着Fボトムプレートで約3 mm、人工血管上で約3 mmであった。血管網構造に関しては、セルカルチャーインサートと人工血管上で作製した融合型肝芽で融合後３日以上維持された（図１A and １F）。
一方で、従来法肝芽を用いた場合、肝芽周囲をマトリクスが覆うことにより肝芽同士の凝集が妨げられ、７日目までにUボトムプレート、Vボトムプレート、セルカルチャーインサート、Fボトムプレート、低吸着Fボトムプレート上での融合が観察されなかった（図2A-E）。Experimental results Aggregation and fusion of human iPSC liver buds
Small liver buds (approximately 80-150 um) prepared on Elplasia plates or conventional liver buds (approximately 1-2 mm) prepared on Matrigel were placed in cell culture inserts, U-bottom plates, and V-bottom plates the next day. , and the aggregation and fusion of liver buds were observed over time up to 7 days. As a result, aggregation and fusion of small liver buds on the cell culture insert, U-bottom plate, V-bottom plate, F-bottom plate, low-adsorption F-bottom plate, and artificial blood vessel were observed from 1 day after fusion (Fig. 1A-F). ). Partial fusion of liver buds was observed in the F bottom plate. The size of the fused liver bud is about 4 mm for the cell culture insert, about 2 mm for the U-bottom plate, about 2 mm for the V-bottom plate, about 3 mm for the low-adsorption F-bottom plate, and about 3 mm for the artificial blood vessel. mm. The vascular network structure was maintained for more than 3 days after fusion between the cell culture insert and the fused liver bud produced on the artificial blood vessel (Figs. 1A and 1F).
On the other hand, when the conventional liver buds were used, aggregation of the liver buds was prevented by covering the surroundings of the liver buds with a matrix. , no fusion was observed on the low-adsorption F bottom plate (Fig. 2A-E).

・融合型肝芽における組織再構築
組織解析により、融合型肝芽においては、直径5-20 μmの毛細血管だけでなく、直径100 μm以上の細小動静脈レベルの血管の再構成が観察された（図３A）。また、肝細胞マーカーのHNF4αやhumanアルブミン陽性の肝細胞クラスターも観察された（図３B and 3C）。蛍光タンパク質クサビラオレンジ導入iPS由来血管内皮細胞を用いたイメージングによっても血管の再構成が確認された（図3D、3E）。共焦点顕微鏡で撮影した画像をImarisにより３Dレンダリングしたところ、血管（赤色）周囲にVimentin陽性のペリサイト様の細胞（黄色）が存在していることが確認された（図3E）。融合型肝芽をクラニアルウィンドウに移植後には、肝細胞に加えて胆管構造も観察された（図3F）。・Tissue reconstruction in confluent hepatic buds Tissue analysis revealed not only capillaries with a diameter of 5-20 μm, but also reconstitution of small arteriovenous vessels with a diameter of 100 μm or more in confluent hepatic buds. (Fig. 3A). In addition, hepatocyte marker HNF4α and human albumin-positive hepatocyte clusters were also observed (Figs. 3B and 3C). Vascular reconstruction was also confirmed by imaging using iPS-derived vascular endothelial cells transfected with the fluorescent protein Kusabira orange (Figs. 3D and 3E). 3D rendering of images taken with a confocal microscope using Imaris confirmed the presence of Vimentin-positive pericyte-like cells (yellow) around blood vessels (red) (Fig. 3E). In addition to hepatocytes, bile duct structures were also observed after implantation of the fused liver buds into the cranial window (Fig. 3F).

・ヒトiPSC融合型肝芽の機能解析
小型肝芽と融合型肝芽それぞれより培養８日目にRNAを回収し、肝分化マーカー計11種類の遺伝子発現解析をqRT-PCR法を用いて行った。その結果、肝分化マーカーであるFoxA2、AFP、CYP3A7、CYP7A1の遺伝子発現レベルが融合型肝芽において有意に上昇していることが明らかとなった（Mann-Whitney’s U-test, p <0.05）（図4）。従来法肝芽とも同様に比較を行い、肝分化マーカーであるALB、AFP、OTC、CYP3A7、GLUT2の遺伝子発現レベルが融合型肝芽において有意に上昇していることが明らかとなった（図５A）。さらに融合型肝芽においてはアルブミン産生能・トランスフェリン産生能、アンモニア代謝が上昇していることが確認された（Mann-Whitney’s U-test, p <0.05）（図５B）。・Functional analysis of human iPSC fusion-type liver buds RNA was collected from each of the small liver buds and fusion-type liver buds on day 8 of culture, and gene expression analysis of a total of 11 types of hepatic differentiation markers was performed using the qRT-PCR method. . As a result, it was revealed that the gene expression levels of FoxA2, AFP, CYP3A7, and CYP7A1, which are hepatic differentiation markers, were significantly elevated in the fused liver buds (Mann-Whitney's U-test, p <0.05) ( Figure 4). A comparison with conventional liver buds was performed in the same manner, and it became clear that the gene expression levels of ALB, AFP, OTC, CYP3A7, and GLUT2, which are hepatic differentiation markers, were significantly elevated in the fusion-type liver buds (Fig. 5A). ). Furthermore, it was confirmed that albumin-producing ability, transferrin-producing ability, and ammonia metabolism were increased in the fused liver bud (Mann-Whitney's U-test, p<0.05) (Fig. 5B).

・ヒトiPSC融合型肝芽のin vivo移植解析
培養4日目の小型肝芽と融合型肝芽を、それぞれマウスクラニアルウィンドウ下に移植を行った（図6A-F）。クラニアルウィンドウ移植法を用いることによって、移植片内の血管内皮細胞とホスト血管の吻合や移植片内の肝前駆細胞の増殖等が、移植動物が生きた状態で経時的に観察可能となる。融合型肝芽移植群では、移植２週間後に30.8%の移植片が生着したのに対し、小型肝芽移植群においては移植片の生着は観察されなかった(それぞれn=13、n=4)（図6A-F）。融合型肝芽移植群においては、融合型肝芽内にあるヒト血管網とホスト血管の吻合と血液灌流が観察され、融合型肝芽内に再構成されたヒト血管網が機能していることが示された（図6G-H）。・In vivo transplantation analysis of human iPSC fusion-type liver buds Small liver buds and fusion-type liver buds on day 4 of culture were transplanted under the mouse cranial window (Fig. 6A-F). By using the cranial window transplantation method, anastomosis between vascular endothelial cells in the graft and host vessels, proliferation of hepatic progenitor cells in the graft, and the like can be observed over time in living transplanted animals. In the fusion-type liver bud transplantation group, 30.8% of the grafts survived two weeks after transplantation, whereas no graft survival was observed in the small liver bud transplantation group (n = 13, n = 13, respectively). 4) (Fig. 6A-F). In the fused liver bud transplantation group, anastomosis and blood perfusion between the human vascular network in the fused liver bud and the host vessel were observed, and the reconstituted human vascular network in the fused liver bud was functioning. was shown (Fig. 6G-H).

・ヒトiPSC融合型肝芽の細胞構成比率の検討
複数の細胞構成比率条件下（iPS-HE:HUVEC:MSC=10:7:1, 10-7:2, 10:7:4, 10:7:7）で融合型肝芽の作製を行った。その結果、MSCの比率が高くなるほど血管網の網目構造が細かくなる傾向が観察された（図7A-D）。この結果より、融合型肝芽内の血管網構造はMSCの比率を変化させることにより制御可能であることが示された。・Examination of the cell composition ratio of human iPSC fusion-type hepatobuds Under multiple cell composition ratio conditions (iPS-HE:HUVEC:MSC=10:7:1, 10-7:2, 10:7:4, 10:7) :7), we prepared fusion-type liver buds. As a result, it was observed that the higher the ratio of MSCs, the finer the network structure of the vascular network (Fig. 7A-D). These results indicated that the vascular network structure within the fused hepatic bud could be controlled by changing the ratio of MSCs.

・ヒトiPSC融合型肝芽のサイズ制御の検討
融合させる小型肝芽の数と融合型肝芽のサイズについて検討を行った。小型肝芽６００個を融合させた場合、融合型肝芽の直径は約4 mmであった（図8A）。小型肝芽１２００個を融合させた場合、融合型肝芽の直径は約6 mmであった（図8B）。小型肝２４００個を融合させた場合、融合型肝芽の直径は約8 mmであった（図8C）。この結果より、融合型肝芽のサイズは、融合させる小型肝芽の数により制御可能なことが明らかとなった。・Examination of size control of human iPSC fusion-type liver buds We investigated the number of small liver buds to be fused and the size of the fusion-type liver buds. When 600 small liver buds were fused, the diameter of the fused liver bud was about 4 mm (Fig. 8A). When 1200 small liver buds were fused, the diameter of the fused liver bud was about 6 mm (Fig. 8B). When 2400 small livers were fused, the diameter of the fused liver bud was about 8 mm (Fig. 8C). These results revealed that the size of the fused liver buds can be controlled by the number of small fused liver buds.

・ヒトiPSC融合型肝芽作製時の細胞塊播種密度検討
小型肝芽の大きさ及び播種密度について検討を行った。直径174 μm、150 μm、137 μmの小型肝芽1200個を21 mm3の細胞接着可能な面に播種したところ、それぞれ播種密度が93%、85%、76%となった（図９）。直径150 μmの小型肝芽600個、300個を21 mm3の細胞接着可能な面に播種したところ、それぞれ播種密度が65%、42%となった（図９）。また、培養９日後には、全ての群において融合型肝芽が得られ、血管形成が確認された（図９）。播種密度が40%未満では均一な細胞塊融合が達成できなかった。・Investigation of cell cluster dissemination density when producing human iPSC fusion-type liver buds The size and dissemination density of small liver buds were examined. When 1200 small hepatic buds with diameters of 174 μm, 150 μm and 137 μm were sown on a surface of 21 mm 3 where cells could adhere, the seeding densities were 93%, 85% and 76%, respectively (Fig. 9). When 600 and 300 small hepatic buds with a diameter of 150 μm were sown on a surface of 21 mm 3 where cells could adhere, the seeding densities were 65% and 42%, respectively (Fig. 9). In addition, after 9 days of culture, fusion-type liver buds were obtained in all groups, and angiogenesis was confirmed (Fig. 9). Uniform cell mass fusion could not be achieved when the seeding density was less than 40%.

・融合型肝芽作製時の細胞比率検討
肝芽材料（HE, EC, MC）の細胞比率の検討を行った。HE:EC:MCの比率をそれぞれ10:7:7, 10:4:4, 10:2:2, 10:1:1, 10:1:2, 10:0.5:2と変化させ、細胞塊を作製した後に上記と同様に高密度に配置した（図１０）。培養９日後に全ての群において融合型肝芽が得られ、血管形成が確認された（図１０）。ECやMCの比率が少なくなるにつれて細い再構成血管が多くなる傾向が観察された（図１０）。MCがEC:MC=1:0.01以下、あるいはECがEC:MC=0.01:1以下では、細胞塊は融合するが血管網の形成は見られなかった。・Investigation of cell ratio at the time of preparation of fusion-type liver bud The cell ratio of the liver bud material (HE, EC, MC) was examined. The ratio of HE:EC:MC was changed to 10:7:7, 10:4:4, 10:2:2, 10:1:1, 10:1:2, 10:0.5:2, respectively, and cell clusters were obtained. were arranged at high density in the same manner as described above (FIG. 10). Fusion-type liver buds were obtained in all groups after 9 days of culture, and angiogenesis was confirmed (Fig. 10). A tendency was observed to increase the number of thin reconstructed vessels as the ratio of ECs and MCs decreased (Fig. 10). When the MC was EC:MC=1:0.01 or less, or the EC was EC:MC=0.01:1 or less, cell clusters were fused, but formation of a vascular network was not observed.

本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

本発明は、ヒューマン・バイオロジー、再生医療、薬剤評価などに利用できる。 INDUSTRIAL APPLICABILITY The present invention can be used in human biology, regenerative medicine, drug evaluation, and the like.

Claims (19)

細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養することを含む、細胞塊融合法であって、細胞塊が器官芽である前記方法。 A cell cluster fusion method comprising seeding cell clusters on a surface capable of cell adhesion and culturing while supplying a medium from the front and back sides of the surface on which the cell clusters are seeded , wherein the cell clusters are organ buds the aforementioned method . 播種面に対して細胞塊が占める面積の比率が４０～１００％となるように細胞塊を細胞接着可能な面上に播種する請求項１記載の方法。 2. The method according to claim 1, wherein the cell aggregates are seeded on the cell-adherable surface so that the ratio of the area occupied by the cell aggregates to the seeding surface is 40 to 100%. 細胞塊が血管細胞及び／又は間葉系細胞を含む請求項１又は２に記載の方法。 3. The method according to claim 1 or 2, wherein the cell mass contains vascular cells and/or mesenchymal cells. 間葉系細胞と血管細胞の割合が１：０．０１～１００である請求項３記載の方法。 The method according to claim 3, wherein the ratio of mesenchymal cells to vascular cells is 1:0.01-100. 間葉系細胞と血管細胞の割合が０．１～１０である請求項４記載の方法。 The method according to claim 4, wherein the ratio of mesenchymal cells to vascular cells is 0.1-10. 融合された細胞塊が血管網構造を形成している請求項１～５のいずれかに記載の方法。 The method according to any one of claims 1 to 5, wherein the fused cell mass forms a vascular network structure. 器官芽が、組織又は臓器細胞、間葉系細胞及び血管細胞から形成されたものである請求項１～６のいずれかに記載の方法。 The method according to any one of claims 1 to 6, wherein the organ buds are formed from tissue or organ cells, mesenchymal cells and vascular cells. 組織又は臓器細胞、間葉系細胞及び血管細胞の割合が、１０：０．１～１０：０．１～１０である請求項７記載の方法。 The method according to claim 7 , wherein the ratio of tissue or organ cells, mesenchymal cells and vascular cells is 10:0.1-10:0.1-10. 間葉系細胞と血管細胞の割合が１：０．１～１０である請求項８記載の方法。 The method according to claim 8 , wherein the ratio of mesenchymal cells to vascular cells is 1:0.1-10. 器官芽が細胞非接着面を有する培養器材中で形成されたものである請求項１～９のいずれかに記載の方法。 10. The method according to any one of claims 1 to 9 , wherein the organ buds are formed in a cultureware having a cell non-adhesive surface. 器官芽が肝芽であり、融合された肝芽が血管網構造を形成している請求項１～１０のいずれかに記載の方法。 The method according to any one of claims 1 to 10 , wherein the organ buds are liver buds, and the fused liver buds form a vascular network structure. 血管網構造が毛細血管及び／又は細小動静脈を含む請求項１１記載の方法。 12. The method of claim 11 , wherein the vascular network comprises capillaries and/or arterioles and veins. 細胞塊が肝芽であり、融合された肝芽が胆管構造を形成している請求項６～１２のいずれかに記載の方法。 The method according to any one of claims 6 to 12 , wherein the cell mass is a liver bud, and the fused liver buds form a bile duct structure. 細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養して、細胞塊を融合させることを含む、融合細胞塊の作製方法。 A method for producing a fused cell cluster, comprising seeding a cell cluster on a surface capable of cell adhesion, culturing while supplying a culture medium from the front side and the back side of the surface on which the cell cluster is seeded, and fusing the cell clusters. 細胞塊を細胞接着可能な面上に播種し、細胞塊を播種した面の表側及び裏側から培地を供給しながら培養し、血管網構造を形成することを含む、血管網構造を構築する方法。 A method for constructing a vascular network structure, comprising seeding cell clusters on a surface capable of cell adhesion and culturing while supplying a medium from the front and back sides of the surface on which the cell clusters are seeded to form a vascular network structure. 肝細胞、間葉系細胞及び血管細胞から形成された肝芽をin vitroで融合することで作製され、血管網構造及び／又は胆管構造を有する、φ４ｍｍ以上の人工臓器。 An artificial organ with a diameter of 4 mm or more , which is produced by in vitro fusion of liver buds formed from hepatocytes, mesenchymal cells and vascular cells, and has a vascular network structure and/or bile duct structure. 肝細胞、間葉系細胞及び血管細胞の少なくとも１つが、人工的に作製された多能性幹細胞から分化誘導されたものである請求項１６記載の人工臓器。 17. The artificial organ according to claim 16 , wherein at least one of hepatocytes, mesenchymal cells and vascular cells is induced to differentiate from artificially produced pluripotent stem cells. φ６ｍｍ以上である請求項１６又は１７に記載の人工臓器。18. The artificial organ according to claim 16 or 17, which has a diameter of 6 mm or more. φ８ｍｍ以上である請求項１６～１８のいずれかに記載の人工臓器。 The artificial organ according to any one of claims 16 to 18, which has a diameter of 8 mm or more .
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